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HSC Cycling Rates

HSC Cycling Rates


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I would like to know how often human hematopoietic stem cells go into cycle in the bone marrow niche (with a paper reference). I have heard they cycle 1-2 times per year but has anyone robustly measured this?


Hematopoietic stem cells: concepts, definitions, and the new reality

Hematopoietic stem cell (HSC) research took hold in the 1950s with the demonstration that intravenously injected bone marrow cells can rescue irradiated mice from lethality by reestablishing blood cell production. Attempts to quantify the cells responsible led to the discovery of serially transplantable, donor-derived, macroscopic, multilineage colonies detectable on the spleen surface 1 to 2 weeks posttransplant. The concept of self-renewing multipotent HSCs was born, but accompanied by perplexing evidence of great variability in the outcomes of HSC self-renewal divisions. The next 60 years saw an explosion in the development and use of more refined tools for assessing the behavior of prospectively purified subsets of hematopoietic cells with blood cell–producing capacity. These developments have led to the formulation of increasingly complex hierarchical models of hematopoiesis and a growing list of intrinsic and extrinsic elements that regulate HSC cycling status, viability, self-renewal, and lineage outputs. More recent examination of these properties in individual, highly purified HSCs and analyses of their perpetuation in clonally generated progeny HSCs have now provided definitive evidence of linearly transmitted heterogeneity in HSC states. These results anticipate the need and use of emerging new technologies to establish models that will accommodate such pluralistic features of HSCs and their control mechanisms.


Translating HSC Niche Biology for Clinical Applications

Over the final 3 decades of the twentieth century, the advent and evolution of hematopoietic stem cell transplantation (HSCT) to cure refractory malignancies and severe non-malignant diseases created a need to better understand basic scientific mechanisms underlying the maintenance and migration of hematopoietic stem cells (HSC). Over the past 20 years, researchers have discovered complex overlapping bone marrow (BM) HSC niches that utilize a number of signaling pathways to tightly regulate HSC physiology. Here, we review recent efforts to define critical mediators of HSC niche function in health and disease, and how these discoveries are now enabling the development of the next generation of cellular therapies for cancer and severe non-malignant diseases.

Recent Findings

A number of cellular interactions and molecular pathways critical for HSC mobilization, donor HSC engraftment after HSCT, and HSC/progenitor cell recovery following chemotherapy have recently been identified. Additional studies have defined mechanisms by which cancer and bone marrow failure states disrupt the normal function of these pathways. Translational investigators are now leveraging these discoveries to develop preclinical and clinical strategies to target the niche for regenerative and cancer therapy.

Summary

Ongoing research to define fundamental aspects of HSC niche biology will lead to further refinements and precision approaches to improve the safety and efficacy of clinical cell therapy.


Biology HSC Syllabus – Maharashtra HSC Board

Higher secondary is the most crucial stage of education because at this juncture specialized disciplines of science are introduced. The present syllabus reinforces the concepts introduced in lower classes. Recently, the science of biology has undergone a paradigm shift that has transformed it from a collection of loosely related facts into a modern applied science.

Living organisms exhibit extremely complex functional system. Organisms seldom occur as isolated individuals. They are organized into populations and biological communities. Organisms, communities, ecosystems and environment constitute unique set of natural resources of great importance. Knowledge of biology helps us to understand a common thread which holds all these components together. Understanding of biology will help in the sustainable development of the environment and will also ensure the existence of earth with all its amazing diversity.

This syllabus is designed to prepare students for various examinations conducted at state and national level. Hence it has been prepared in accordance with the guidelines shown in the final version of common core syllabi of COBSE, Delhi. Accordingly some additional topics from state Board syllabus have been deleted whereas the lacking topics have been added. The entire unit “Ecology and Environment” has now been added under Botany and Zoology sections.

  • The prescribed syllabus is expected to
  • Promote the inherent skill of observation.
  • Assist to understand the underlying principles of biological sciences and thereby develop scientific attitude towards biological phenomena.
  • Help students to understand the functioning of organisms.
  • Make students aware of issues of global importance.
  • Guide students to perform easy experiments for better understanding of biological principles and to develop experimental skills required in practical work.
  • Create awareness about the contribution of biology to human welfare

Section I – BOTANY

Unit 1: Genetics and Evolution:

Chapter 1 – Genetic Basis of Inheritance:

Mendelian inheritance. Deviations from Mendelian ratio (gene interaction-incomplete dominance, co-dominance, multiple alleles and Inheritance of blood groups), Pleiotropy, Elementary idea of polygenic inheritance.

Chapter 2 – Gene: its nature, expression and regulation:

Modern concept of gene in brief-cistron, muton and recon.

DNA as genetic material, structure of DNA as given by Watson and Crick’s model, DNA.

Packaging, semi conservative replication of eukaryotic DNA

RNA: General structure types and functions.

Protein Synthesis central dogma, Transcription Translation-Genetic Code, Gene Expression and Gene Regulation (The Lac operon as a typical model of gene regulation).

Unit 2: Biotechnology and its application:

Chapter 3 – Biotechnology: Process and Application :

Genetic engineering (Recombinant DNA technology):

Transposons, Plasmids, Bacteriophages

Producing Restriction Fragments, Preparing and cloning a DNA Library,

Gene Amplification (PCR). Application of Biotechnology in Agriculture – BT crops Biosafety Issues (Biopiracy and patents)

Unit 3: Biology and Human Welfare:

Chapter 4 – Enhancement in Food Production

Plant Breeding Tissue Culture: Concept of Cellular

Totipotency, Requirements of Tissue Culture (in brief),

Callus Culture, Suspension Culture.

Single Cell Protein. Biofortification.

Chapter 5 – Microbes in Human Welfare:

Microbes in Household food processing.

Microbes in Industrial Production.

Microbes in Sewage Treatment.

Microbes in Biogas (energy) Production.

Microbes as Biocontrol Agents Microbes as Biofertilizers.

Unit 4: Plant Physiology:

Chapter 6 – Photosynthesis

Autotrophic nutrition Site of Photosynthesis Photosynthetic Pigments and their role.

Light-Dependent Reactions (Cyclic and non-cyclic photophosphorylation)

Light-Independent Reactions (C3 and C4 Pathways) Chemiosmotic hypothesis, Photorespiration, Factors affecting Photosynthesis. Law of limiting factors.

Chapter 7 – Respiration

ATP as currency of Energy Mechanism of Aerobic (Glycol sis, TCA Cycle and Electron Transport System) and Anaerobic Respiration. Fermentation Exchange of gases. Amphibolic pathway. Respiratory quotient of Nutrients. Significance of Respiration.

Unit 5: Reproduction in Organisms:

Chapter 8 – Reproduction in Plants

Modes of Reproduction (Asexual and Sexual).

Asexual reproduction uniparental modes vegetative propagation, micro propagation Sexual Reproduction: structure of flower Development of male gametophyte, Structure of anatropous ovule. Development of female Gametophyte. Pollination: Types and Agencies. Outbreeding devices pollen-pistil interaction. Double Fertilization: Process and Significance. Post-fertilization changes (development of endosperm and embryo, development of seed and formation of fruit) Special modes-apomixis, parthenocarpy, polyembryony. Significance of seed and fruit formation.

Unit 6: Ecology and Environment

Chapter 9: Organisms and Environment -I : Habitat and Niche

Ecosystems: Patterns, components, productivity and decomposition, energy flow pyramids of number, biomass, energy nutrient cycling (carbon and phosphorous). Ecological succession, Ecological services carbon fixation, pollination, oxygen release. Environmental issues: agrochemicals and their effects, solid waste management, Green house effect and global warming, ozone depletion, deforestation, case studies (any two).

Class 12th Biology

Section II – ZOOLOGY

Unit 1: Genetics and Evolution:

Chapter 10 – Origin and the Evolution of Life:

Origin of Life: Early Earth, Spontaneous, assembly of organic compounds, Evolution: Darwin’s contribution, Modern Synthetic Theory of evolution, Biological Evidences, Mechanism of evolution Gene flow and genetic drift Hardy Weinberg principle Adaptive radiation. Origin and Evolution of Human being.

Chapter 11 – Chromosomal Basis of Inheritance

The Chromosomal Theory. Chromosomes. Linkage and Crossing Over. Sex-linked Inheritance (Haemophilia and color blindness). Sex Determination in Human being, birds, honey bee. Mendelian disorders in humans-Thalassemia. Chromosomal disorders in human: Down’s syndrome, Turner’s syndrome and Klinfelter’s syndrome.

Unit 2: Biotechnology and its application:

Chapter 12- Genetic Engineering and Genomics

DNA Finger Printing. Genomics and Human Genome Project. Biotechnological Applications in Health: Human insulin and vaccine production, Gene Therapy. Transgenic animals.

Unit 3: Biology and Human Welfare

Chapter 13- Human Health and Diseases

Concepts of Immunology: Immunity Types,

Structure of Antibody, Antigen-Antibody Complex, Antigens on blood cells. Pathogens and Parasites (Amoebiasis, Malaria, Filariasis, Ascariasis, Typhoid, Pneumonia, Common cold and ring worm). Adolescence, drug and alcohol abuse. Cancer and AIDS.

Chapter 14- Animal Husbandry

Management of Farms and Farm Animals.

Unit 4: Human Physiology :

Chapter 15- Circulation

Blood composition and coagulation, Blood groups.

Structure and pumping action of Heart. Blood Vessels.

Pulmonary and Systemic Circulation.

Heart beat and Pulse. Rhythmicity of Heart beat. Cardiac output, Regulation of cardiac activity.

Blood related disorders: Hypertension, coronary artery disease, angina pectoris, and heart failure. ECG, Lymphatic System (Brief idea): Composition of lymph and its functions.

Chapter 16- Excretion and osmoregulation

Modes of excretion-Ammonotelism, ureotelism, uricotelism. Excretory System. Composition and formation of urine. Role of Kidney in Osmoregulation.

Regulation of kidney function: reninangiotensin, atrial natriuretic factor, ADH and Diabetes inspidus, role of other organs in excretion.

Disorders Kidney failure, Dialysis, Kidney stone (renal calculi). Transplantation. Uraemia, nephritis.

Chapter 17- Control and Co-ordination

Nervous System Structure and functions of brain and Spinal cord, brief idea about PNS and ANS. Transmission of nerve impulse. Reflex action. Sensory receptors (eye and ear), Sensory perception, general idea of other sense organs.

Hormones and their functions

Mechanism of hormone action.

Hormones as messengers and regulators.

Hormonal imbalance and diseases:

Common disorders (Dwarfism, Acromegaly, cretinism, goiter, exopthalmic goiter, Diabetes mellitus, Addison’s disease)

Unit 5: Reproduction in Organisms :

Chapter 18- Human Reproduction

Reproductive system in male and female.

Histology of testis and ovary.

Production of gametes, fertilization, implantation.

Embryo development up to three germinal layers.

Pregnancy, placenta, parturition and Lactation (Elementary idea).

Reproductive health-birth control,

Contraception and sexually transmitted diseases.

MTP, Amniocentesis Infertility and assisted reproductive Technologies IVF, ZIFT, GIFT (elementary idea for general awareness).

Unit 6: Ecology and Environment:

Chapter 19-Organisms and Environment-II :

Population and ecological adaptations:

Population interactions-mutualism, competition, predation, parasitism, Population attributes- growth, birth rate and death rate, age distribution.

Biodiversity and its conservation Biodiversity- concept, patterns, importance, loss.

Threats to and need for biodiversity conservation, Hotspots, endangered Organisms, extinction, red data book, biosphere reserves, national parks and sanctuaries.

Environmental issues: air pollution and its control, water pollution and its control and radioactive waste management. (Case studies any two)

(Upgraded) Biology Practical’s Experiments

  1. Dissect the given flower and display different whorls. Dissect anther and ovary to show number of chambers.
  2. Study pollen germination on a slide.
  3. Collect and study soil from at least two different sites and study them for texture, moisture content, pH and water holding capacity of soil. Correlate with the kinds of plants found in them.
  4. Study of plant population density and frequency by quadrat method.
  5. Prepare a temporary mount of onion root tip to study mitosis.
  6. Separation of plant pigments by paper chromatography.
  7. A) To study the rate of respiration in flower buds/leaf tissue and germinating seeds.
  8. B) Demonstration of anaerobic respiration.
  9. Study the presence of suspended particulate matter in air at the two widely different Sites.
  10. Collect water from two different water bodies around you and study them for pH, clarity and presence of any living organisms.
  11. To test the presence of urea and sugar in urine.
  12. To test the presence of albumin and bile salts in urine.

Study/observation of the following (Spotting):

  1. Study of flowers adapted to pollination by different agencies (wind, insect)
  2. Study of pollen germination on stigma through a permanent slide.
  3. To Study Mendelian inheritance using seeds of different color/size of any plant.
  4. Exercise on controlled pollination – Emasculation, tagging and bagging.
  5. Study meiosis in onion bud cell or grass hopper testis through permanent slides.
  6. Study of plants found in xerophytic and aquatic conditions with respect to their morphological adaptations.(Two plants each)
  7. Study and identify stages of gamete development, i.e. T.S. of testis and T.S. ovary through permanent slides (from any mammal).
  8. Study of V.S. of blastula through permanent slide.
  9. To study prepared pedigree charts of genetic traits such as rolling of tongue, Blood groups, widow’s peak, color blindness.
  10. To identify common disease causing organisms like Plasmodium, Entamoeba, Ascaris and ring worm through permanent slides or specimens. Comment on symptoms of diseases that they cause.
  11. Study of animals found in xeric (desert) and aquatic conditions with respect to their morphological adaptations. (Two animals each)

Note: We make no claims on the accuracy and reliability of the information. For correct/current information kindly contact the concerned authorities.


Module 5 / Inquiry Question 1

Before we hop on the materialistic train and start digging into the content, please give me a minute to walk you through what you should keep in mind as the major highlights for this week’s material.

The inquiry (overarching) question for this week deals with reproduction and its relationship with evolution, aka. the continuity of species.

Under the concept of reproduction, we are specifically concerned about the reproductive mechanisms (how they work) occurring in animals, plants, fungi, bacteria and protists.

We need to classify reproductive processes as sexual or asexual on top of how their mechanisms that allow parent(s) producing and passing genetic materials onto their offsprings.

Out of all types of species, NESA wants us to dive into the land of mammals (e.g. reindeer and human) and explore how their reproduction systems work.

We need to understand the process of fertilisation, implantation and hormonal control during reproduction. These stages of reproduction help pass on genetics materials from parent(s) to their offspring.

I am not sure if you are aware but humanity’s scientific knowledge has advanced a lot over the past century.

So, in the last part of this week’s material, we will turn to some real life application examples of humans applying scientific knowledge in genetics and reproduction to create AWESOME variations in plants and animals!

Learning Objective: Explain the mechanisms of reproduction that ensure the continuity of a species, by analysing sexual and asexual methods of reproduction in a variety of organisms.

Did you not knowing or writing out a definition for the main keyword can cost you a mark in HSC Questions?

Let’s first define reproduction!

Reproduction is the process of creating a new individual or offspring from their parent(s).

Reproduction means to reproduce = new offspring. They MAY or MAY NOT be clones of the parent.

Reproduction can be done via natural or artificial means. Hence, the terms natural and artificial reproduction.

There are two reproductive pathways: sexual and asexual. Some organisms can do both!

What are sexual and asexual reproduction and how do they work?

Sexual reproduction: The process of forming a new organism from the fusion of the offspring’s parents’ (male + female) gametes. Gametes are sex cells such as sperm and egg cells for humans. The offspring that is formed from sexual reproduction has the genetic material that is derived from its parents. However, in almost* all cases the offspring’s genetic material is NOT IDENTICAL to their parents (it’s mixed). In humans and many other mammals such as cows this process of producing gametes is called meiosis .

*Note that self pollination involves one plant (parent) and is a type of sexual reproduction. This is because the plant can produce both pollen and ovules (male and female plant gametes). These gametes can combine to produce either a genetically identical offspring or genetically different offspring. Whether the offspring is genetically identical or different, it will depend of whether the single parent plant is homozygous or heterozygous for those genes. We will learn about these two terms when you learn about Punnett Square in Week 4’s notes.

Asexual reproduction: Asexual reproduction is the process of forming an offspring (usually a cell) from just ONE parent through cell division. Depending on the cell division process, there may be many names. For example, in humans and many other mammals, this cell division process is called mitosis . Thus, the offspring has genetic materials that is IDENTICAL to that of its single parent – offspring is a CLONE of the parent.

The most important distinguishing factor between sexual and asexual reproduction is whether or not the fusion of gametes occurred. For sexual reproduction, there must be a fusion of gametes whereas, in asexual reproduction, there is no fusion of gametes.

How sexual and asexual reproduction processes allow parents’ genetic information to be passed on to their offspring, and thus, ensuring the continuity of the species?

During reproduction, the parents’ genetic information (DNA) is copied and passed onto the offspring. The offspring’s genetic material is stored in their cells’ nucleus.

There are two types of cells: somatic and non-somatic (sex) cells. Since humans and many other mammals cannot produce offspring via asexual means, all offsprings produced are from non-somatic cells. Hence, only the parents’ genetic material in non-somatic cells’ (or sex cells’) genetic material is passed onto the offspring.

Genetic information is passed onto the next generation (offspring). Thus, ensuring the continuity of the species.

By week 3’s notes, you will see the importance of creating variation in offspring’s genetic information (new allele combinations, increasing variation in the alleles which gametes can inherit as well as variation in the gametes that are fertilised during fertilisation). Don’t worry if you don’t know what genes and alleles are, we will look into their definitions and role in genetics in Week 2. Essentially, the point is that you will see how the increased variation in the offspring’s genotype will enhance the chances of survival of a species’ population and thus supporting the continuity of the species. In Week 4’s notes, you will see how mutation (apart from sexual and asexual reproduction) can also create genetic variation.

A third point is that reproduction would increase the total number of offsprings in a population, effectively increasing population size. Thus, supporting the continuity of species.

What is evolution?

Evolution is the change in living organism’s genetic information, favourable characteristics and phenotypes (appearance or physical traits) over many generations.

For now, just know that genetic information contributes to an organism’s phenotype. In terms of how this works, this will be comprehensively covered in the upcoming weeks.

But what drives evolution aka. the change in genetic information, for example, how a rainbow unicorn population can slowly turn into a pack of green unicorns, over time?

How and where does reproduction fit in Darwin’s Theory of Evolution by Natural Selection?

Darwin’s Theory of Evolution by Natural Selection is a popular and widely accepted modern theory of evolution.

It explains the drivers and consequences of evolution to a reasonable extent.

It, however, fails to account the origins of life on Earth or any place in our universe from start to finish.

There are other models that deals with the origin of life and such as the RNA world hypothesis but they are not complete.

Please keep in mind that these are all theories! Yes, there are evidence to back them up. However, existing evidence is not complete to transform these theories into universal laws!

In order, here are the Stages of Darwin’s Theory of Evolution by Natural Selection:

1. There is genetic variation in population, which affects it phenotype (physical traits). The genetic variation is derived from a number of factors – from internal biological processes to external environmental factors. These factors will be comprehensively covered in later weeks.

2. The majority of the existing population would have the favourable traits that allow them to survive in the environmental conditions (temperature, food supply, predators, etc) that they are exposed to.

3. There is a SUDDEN change in environmental conditions (e.g. new predator introduced to kill the unicorns, sudden large drop in temperature, a virus, etc)

4. Those organisms with favourable characteristics, derived from favourable genes passed on from parents, will survive and those with less or without favourable characteristics will decline in numbers.

5. Those organisms with favourable characteristics will reproduce more successfully and pass on their favourable genetic information to their offspring. REPRODUCTION FITS IN HERE!

6. Over time, the new population will predominately be made up of organisms with favourable characteristics that allow them to tolerate the new environmental conditions.

The environmental agent is refers to the environmental change. This could be a exotic species introduced into the habitat (e.g. from migration) that is competing for the same food resource as the existing population, a new predator, introduction of chemicals into the environment – e.g. toxic wastes being throw into the river, home to thousands of fish..

It is called Darwin’s Theory of Evolution by Natural Selection because there is a sudden change is due to environmental (nature) change(s).

What are favourable characteristics in Darwin’s Theory of Evolution?

How can organisms acquire these awesome characteristics?

Favourable characteristics that allow organisms to survive in their environment can take three forms:

Physical, Physiological and Behavioural.

‘Favourable’ means that these characteristics allow the organism to specifically or better cope with its ambient environment.

As these characteristics are derived from genetic material INHERITED over generations, they are also referred to as adaptations.

For HSC purposes, an organism CANNOT adapt to its environment during its lifetime.

Adaptations are inherited.

Example: A snake cannot learn to seek shade to prevent itself from overheating during its lifetime if it did not inherit such behavioural characteristics from its parents.

However, you will learn later in Module 6 that a mutation can also give rise to adaptation.

The following characteristics or adaptations are evolved through many generations:

Physical characteristics (PHENOTYPE): Large ears to facilitate cooling. This is favourable for organisms living in hot environments.

Physiological characteristics: Kangaroos licking their paws to encourage evaporation and cooling down. Favourable in hot environments.

Behavioural characteristics: Snakes hiding under rocks to avoid the sun. Favourable in hot environments or during the middle of the day.

In Step 1 of Darwin’s Theory of Evolution by Natural Selection, it was mentioned that there is genetic variation in the population. The main sources of variation are:

Mutation of DNA as a result of environmental factors

DNA replication error during meiosis

Independent assortment and random segregation during meiosis

We will go into details of these sources of variation in the later weeks.

For now, just understand where these factors fit in within the areas of evolution and reproduction that we covered so far.

NOTE: It is important to note that asexual reproduction does not introduce genetic variation in offspring while sexual reproduction does. Despite this, the parent of the offspring has favourable characteristics (adaptations) to allow the parent to tolerate the selective pressures of the ambient environment, asexual reproduction allows the parent to produce offspring with IDENTICAL genetic information (no genetic variation) that codes for the same favourable characteristics (e.g. long or short ears depending on environment temperature). The offspring will now have the same favourable characteristics as parent due to inheriting identical genetic information and thus have the same survival rate of parent if they are exposed to same environment with the same resources.

RECAP on what we covered so far,

Two categories of reproduction that can take place (sexual & asexual)

Reproduction allows genetic information to be passed on to offspring via heredity, ensuring continuity of species

Reproduction increases population size thus supporting the continuity of species

Genetic variation helps increase the population’s overall survival rate and thus support the continuity of species.

Sexual and asexual reproduction are both useful in supporting the continuity of species despite asexual reproduction not introducing genetic variation in offspring and, thus, population.

Reproduction plays an important role during Darwin’s Theory of Evolution via Natural Selection

We will now explore the SPECIFIC TYPES OF REPRODUCTION in the sexual and asexual reproduction categories!

This is very eggciting! Get the joke? Sorry, I needed to do it.

Analyse whether the types of reproduction methods are sexual or asexual?

How do they work?

Internal fertilisation vs External Fertilisation

Internal fertilisation involves the fusion of male and female gametes within a parent’s body. Internal fertilisation tends to occur between terrestrial animals.

External fertilisation involves the fusion of male and female gametes outside a parent’s body. External fertilisation tends to occur between aquatic animals.

Parthenogenesis in animals

Parthenogenesis is the process whereby an unfertilised egg develops into an functional offspring. This is a form of asexual reproduction in animals, e.g. bees. For bees, queen bees can produce egg cells (gametes) via meiosis. These egg cells can undergo parthenogenesis to produce haploid drone (male) bees. Haploid cells are cells that have half the amount of chromosome as parent. Chromosomes contain DNA which you will explore in Week 2 notes. Usually parthenogenesis occurs due to the organism’s hardship in having access to mating partners. This is common for organisms residing in harsh or extreme environments. For the most part, the haploid cell develops as it would it would a diploid cell. So, essentially, the gamete undergoes mitosis to develop into a drone bee which will have a diploid chromosome number.

Plants can also undergo parthenogenesis which is called apomixis.

Mechanisms of Cross-Pollination vs Self-Pollination

Cross pollination involves the transfer of pollen, produced by anther (which is part of the plant’s stamen), to the stigma of another plant. This means that cross pollination involves two plants. The pollen grain essentially contain the male gametes of the plant. Bees, wind and water can be transport methods of pollens grain to stigma of another plant for cross pollination. Pollination is referred to the process where the pollen is successfully transfered to the stigma of another plant.

Once the pollen is on the stigma, it can grow a pollen tube which runs down the style of the plant and eventually into the ovary of the plant which produces the ovules which contains female gametes (ovum or ova) of the plant. Fertilisation occurs inside the ovule where the pollen can fertilise the ovule where male gametes are combined with the ovum inside the ovule forming a zygote. The zygote is diploid, i.e. has double the chromosomes of each of the male and female gametes which are both haploid. We will discuss more of diploid and haploid when we explore Mitosis and Meiosis next week.

Note that most pollen grains contain two male gametes. One fertilises the ovum inside the ovule and the other male gamete fertilises two polar nuclei (diploid nucleus) inside the ovule which develops into a endosperm which is a tissue that supply nutrients to the zygote (seed) when it grows.

Fun fact: This means that the endosperm nucleus is a triploid (contains three sets of homologous chromosomes or three copies of each chromosome). Humans are diploids (we contain two sets of chromosomes, i.e. two copies of each chromosome).

Note that chromosomes in the homologous sets are not necessarily identical copies as the chromosomes may contain different alleles for the same gene. We will explore more about alleles next week.

This fertilised ovule is called a seed which contains the zygote and will develop into an embryo. In some plants, the surrounding space of the ovule will develop into a fruit. Other plants such as sunflowers do not form fruit, what happens is that the seed will drop from the original sunflower which will develop into another sunflower when the seed germinates under the favourable conditions. The seed will germinate (grow) into a plant via mitosis. In some other flora, the ovary will become a fruit. However, this is not for sunflowers as they do not grow fruits XD.

It is important to note that most plants have its own stigma and stamen. Self-pollination is similar to cross pollination. The difference between self-pollination and cross-pollination is that self-pollination does NOT involve a an external agent such as bees, water and wind as mentioned previously. Instead, the stigma can reshape itself to enclose the stamen. This means that the pollen can be easily transferred onto the stigma.

It is important to note that self pollination causes the resulting flower offspring (after seed germination) to have far less genetic variation than their parents in most cases compared to cross pollination. This is because the resulting flower is only produced from only one parent plant rather than two in cross pollination. If the parent in self-pollination is heterozygous for some genes, the resulting flower may have probabilities of being genetically different to their parents for those genes. We will examine why this is the case when we do Punnett Squares in Week 4 where we learn about homozygous and heterozygous alleles for different genes.

Cross pollination will result in a sunflower offspring that genetically different to its parents. It involves the transfer of pollen from one plant to the stigma of a different plant.

Vegetative Propagation

You may have heard of vegetative propagation at school. How does vegetative propagation fit in all of this?

Well, vegetative propagation is a type of asexual reproduction that occurs in plants. It results in the parent producing a plant that is genetically identical. Runners, bulbs, fragmentation are some examples of vegetative propagation. Let’s have a look at them now.

Fragmentation

Fragmentation is when the original organism separates a small part of itself. This occurs in starfish where a part of its body can be separated from its parent and the separated section can develop into a new starfish that is genetically identical to parent starfish via cell division.

Fragmentation can also occur in mosses when you split one moss into two. The moss will grow via cell division when it becomes into contact with matter such as moisture in the air.

Runners

Strawberry plants can develop runners which are stems extending from the plant and along the soil. At certain points along the runners, nodes can develop which extends to the soil, resulting in the formation of new plant roots at another area of the soil whereby a new strawberry plant can grow. The runner joins the new (and genetically identical) strawberry plant to the parent plant.

Bulbs

Bulbs are bud cells that are found underground. These buds can develop into new plants such as onions. When a new plant forms, the underground bulb provide nutrients to the plant for its survival.

Budding in Fungi

Budding in fungi such as yeast involves the parent cell developing a bud cell, a daughter nucleus. This usually occur when the environmental conditions are favourable for the fungi. Over time, this bud undergoes cell division while still being attached to the parent which may result in a chain of bud cells due to cell division. During cell division, but prior to separation of the protruding bud from the parent yeast (fungi), the parent’s nucleus’ DNA replicates and nucleus divides equally, but, the cytoplasm divides unequally (hence bud is smaller than parent). One copy of the DNA moves into the bud cell which results in the successful transfer of the parent’s DNA into the daughter (bud) cell. The bud separates from its parent fungus when it grows to a sufficient size to be able support itself independently. This now-separated bud undergoes further cell division to produce more bud cells. The result is yeast that is genetically identical to parent.

Budding is also found in another type of organism called Hydras and the budding process is similar to that of fungi.

Asexual spore production in Fungi

Spores are microscopic reproductive units (cells) that can be formed as a result of mitosis or meiosis.

Spores different to gametes as they do NOT need to combine or be fertilised by another spore to form an offspring.

Mycelium is part of a fungi that branches out into a network structure of fine ‘threads’ called hyphae (plural for hypha). Each hypha have ends of that are capable of producing spores called sporangia (plural for sporangium). These sporangia (and thus spores) are produced when environment conditions are favourable for the fungi’s survival. Mushroom is a type of fungi where the mushroom cap is above the hyphae spread along the stem and to the mushroom cap. The mushroom cap therefore has basida, which are examples of sporangia, that produces spores.

These asexual spores are usually produced when ambient environment conditions are favourable via mitosis. These spores are usually carried by the wind as they are light-weight. These spores then germinates to form genetically identical fungus when environmental conditions are favourable. This typically involves the spores absorbing moisture and decaying organic matter from its environment, allowing the cytoplasm to expand and the fungus developing into a mycelium whereas new spores can be produced.

Sexual spore production in Fungi

Sexual spores are developed when opposite gender hyphae are combined together to develop a sporing-producing structure known as zygospore. The zygospore is diploid as each of the hypha are haploid. Under favourable conditions, the diploid zygospore undergoes meiosis to produce haploid sexual spores which are dispersed into the environment. These spores that are genetically different from their parents.

Under favourable conditions, these spores will germinate and a genetically different fungus to its parents will be formed. These fungi are haploid as most fungi spend their lives as haploid organisms until time of sexual reproduction where hyphae combine to form a diploid zygospore to produce haploid sexual spores.

In some fungus, the mycelium contains hyphae of two genders (male and female). This means that these fungus can produce spores via meiosis and disperse them into the environment.

The term ‘plasmogamy’ refers event where the nucleus of the one hyphae enters the cytoplasm of another hyphae.

The term ‘karyogamy’ refers to the event where the two nucleus are combined into one.

Binary fission in Bacteria

Binary fission is most commonly performed by unicellular organisms such as bacteria, though some multiceullar organisms can reproduce asexually via binary fission too. The process starts with the copying the genetic material (in the form of bacterial chromosomes) of the parent cell. Each chromosome moves to each side of the cell. This is followed by the elongation of the cell and cytokinesis which is the splitting of the cell membrane and cytoplasm of the cell into two daughter cells. As there is no cell nucleus in bacteria, there will not be the splitting of cell nucleus. It is important to note that the parent cell won’t exist at the end because it is now part of the two daughter cells. The two daughter cells are genetically identical to each other as well as identical to the parent which they obtained their genetic information came from.

NOTE: There are multicellular organism that reproduce asexually via binary fission. However, they are uncommon. Some example of this is the organism named Trichoplax.

Budding in Protists

Budding in protists is a type of asexual reproduction. In short, budding in protists starts off by the parent protozoan producing a bud which is a daughter nucleus that is created based on the replicate of nucleus DNA, followed by equal nucleus division but unequal separation of the parent protozoan’s cytoplasm. This means that the bud is smaller than the parent. Over time, this daughter nucleus undergoes further cell division via mitosis to grow and mature, resulting in a protists that is genetically ideal to parent.

Binary fission in Protists

The mechanism of binary fission in protist is similar to that of bacteria’s binary fission process. However, as DNA is stored in the nucleus (whereas no nucleus in bacteria), the chromosome will move to each side of the nucleus before the splitting of the nucleus and eventually splitting of the cell membrane and cytoplasm into two daughter cells. The splitting of the parent cell into two daughter cells is called cytokinesis.

NOTE: Binary fission in protist vs bacteria and budding in protist vs fungi are similar. So, it is important to determine the unique characteristics fungi and protist.

Protists are mostly unicellular whereas fungi are mostly multicellular.

Protists are microscopic whereas fungi are macroscopic.

Protists are eukaryotes whereas bacteria are prokaryotes.

Advantages and disadvantages of internal and external fertilisation

Internal fertilisation

• Internal fertilisation occurs inside the female’s body which means that the zygote is protected from the external environment of the parent. This means there are less environmental factors that affect the zygote in internal fertilisation compared to external fertilisation. This increases the survival of the zygote.

• Internal fertilisation is NOT restricted to terrestrial environments unlike external fertilisation which is restricted to aquatic environments only.

• Internal fertilisation has higher fertilisation success rate on a per gamete basis compared to external fertilisation. This is because the sperm does not need to travel by chance to fertilise an egg. Internal fertilisation provides the sperm a direct route towards to egg cell inside the female’s body. During such journey, the sperm cell is subjected to less variable and/or violet environment factors such as strong current or predators.

Disadvantages:

• Internal fertilisation typically have less mating partner options than external fertilisation. This can lead to a lower genetic variation in species population as the mating process is more selective than external fertilisation

• Internal fertilisation generally required more energy in search for a mating partner and perform the mating process which are unnecessary in external fertilisation.

• Less gametes are produced via internal fertilisation compared to external fertilisation. This leads to a lower overall amount of offsprings produced. Arguably, this means that internal fertilisation may low the chance of the continuity of a species (if we assume that genetic variation is controlled for both internal and external fertilisation, i.e. genetic variation is the same for both external and internal fertilisation).

External Fertilisation

• Greater quantity of gametes are produced via external fertilisation compared to internal fertilisation. This leads to a greater overall amount of offsprings produced. Arguably, this could supports the continuity of species more than internal fertilisation.

• External fertilisation can give raise to more mating partner options than internal fertilisation. This can lead to greater genetic variation in species population as the mating process is less selective than internal fertilisation.

Disadvantages:

• Upon fertilisation, the zygote is exposed to the environment rather than protected inside the mother’s body for internal fertilisation. Due to the limited defence capabilities of the zygote (e.g. against predators), it is more susceptible to death than zygotes found via internal fertilisation. Most of the gametes are being attacked by predators or fail to be fertilised. The zygote therefore has a lower chance of survival via external fertilisation.

External fertilisation is restricted to aquatic environments. The flagellum component of the sperm cell allows it to move through water that otherwise would not be possible on land. If performed on land, the egg will dry out.

External fertilisation has a lower fertilisation success rate than internal fertilisation. This is because the sperm and egg cells are subjected to greater amount of factors in external fertilisation than internal fertilisation. For example, the more environmental factors such as predators (Sea life) and harsh aquatic environment conditions (e.g. harsh currents).

Example of a case of external fertilisation (Sea urchin):

Male and female sea urchins produce gametes which are dispersed in the ocean.

Male salmon produce gametes (Sperm) to fertilise a nest of eggs that is produced by female salmon somewhere in the ocean.

Extra Notes on sexual and asexual reproduction

Now that we have explored asexual and sexual reproduction with examples, let’s see what they involve beyond differences between number of parents involved and genetic variation in offspring that we have mentioned at the beginning of this notes.

Here are some extra notes between sexual and asexual reproduction:

Sexual reproduction requires more energy than asexual reproduction.

However, asexual reproduction tends to occur at a faster rate than sexual reproduction.

Genetic variation is created in sexual reproduction and NOT in asexual reproduction.

Genetic variation increases the likelihood of the continuity and evolution of the species – relating back to inquiry question.

Asexual reproduction would also be a concern if the parent genes code an unfavourable trait because there is no other source of genes from another parent to override it.

This problem is reduced in sexual reproduction as the offspring’s genome is a mix of both parents (rather than single parent) and unfavourable trait could be overridden.

More details about overriding genes in later weeks. It is based on concepts of dominant and recessive genes.

Asexual reproduction generally ONLY take place because the ambient environment conditions are favourable as asexual reproduction does not increase variability in genetic materials.

An asexual offspring is a clone of its parent. If one clone is affected, the whole cloned population have equally as great of a danger for extinction.

Well done! we have broadly covered reproduction processes for a range of organisms. We will now examine reproduction for mammals specifically!

Learning Objective: Analyse the features of fertilisation, implantation and hormonal control of pregnancy and birth in mammals

Fertilisation

Requires gametes (sperm and egg) meet and combine to form a zygote

Gametogenesis is the name of the gamete formation process.

Gametogenesis can be divided into spermatogenesis (producing sperm) and oogenesis (formation of matured egg cells)

The hormone testosterone is produced in cells’ in the testes organ of male as part of spermatogenesis as it plays a role in producing sperm cells.

The hormone oestrogen in males help with the maturing of the sperm cells in males.

The fertilisation process and fusion of gametes occurs in the fallopian tube of female’s body

The zygote will develop into a living organism that has mixed genetic information from the parents.

Zygote is the continuity of a species (relating back to inquiry question)

Fertilisation involved multiple stages that MUST be fulfilled for successful fertilisation and zygote formation and thus producing a new offspring.

Three necessary stages for successful fertilisation are:

Formation and maturation of gametes

Spermatozoa must journey into the oviduct

Spermatozoa must make contact and fuse with the egg cells.

The gametes fuse with one purpose – to form a zygote, single cell with 46 chromosomes

During fusion, the head of the sperm cell detaches from its tail (flagellum) and the sperm-egg species journeys down the female’s uterus.

Also, during fusion, the sperm cell activates the egg cell resulting in cell division of the egg cell growth/development. The resulting product is called a blastocyst.

Once the sperm fused with the egg, other sperms will no longer be able to fuse with the same egg

Most of our contemporary knowledge of fertilisation in mammals comes from laboratory testing with mice gametes.

The gametes must be from the same species in other for successful fertilisation.

Implantation

Implantation is the process of adhering the fertilised egg to stick to the walls of the reproductive tract, providing the most suitable environment for zygote development.

It is a crucial phase for successful pregnancy.

The blastocyst is implanted on the walls of the reproductive tract (uterine wall).

Successfully implantation means pregnancy.

This implantation process onto the walls establishes blastocyst’s access to nutrients to develop into an embryo (blood vessels surrounding the blastocyst carries blood which has dissolved nutrients)

Embyro develops into a fetus (approx 5-11 weeks)

Embryro becomes a new organism upon release from female’s body.

The bottom left image is diagram showcasing the steps of fertilisation and implantation:

The idea of the diagram is just to allow you have a rough idea of where fertilisation and implantation occurs in the female’s body. The steps in the diagram not as important.

Note, at ovulation stage, the matured egg cell is released from the follicle and travels up and along the fallopian tube (the C-shaped tube as shown in diagram below) that connects the ovary to the uterus. It at the uterus where the embryo is implanted on the uterus wall (endometrium) during implantation phase.

Successful implantation of the embryo means successful pregnancy.

Note that: When the sperm enters the vagina, up to the uterus, along and down the fallopian tube where it can combine and fertilise the mature egg. This means that the mature egg and sperm encounter each other head-on as the egg is moving in the direction from ovary to uterus and sperm is moving in the direction of uterus to ovary.

This means that they are likely to meet at the fallopian tube, which is where fertilisation of the mature egg cell most commonly takes place in reality.

In the diagram below, we see that the zygote (fertilised egg) is formed in the fallopian tube where the sperm meets and fertilises the egg.


Cell cycle regulation in hematopoietic stem cells

E.M. Pietras and M.R. Warr contributed equally to this paper.

Eric M. Pietras, Matthew R. Warr, Emmanuelle Passegué Cell cycle regulation in hematopoietic stem cells. J Cell Biol 28 November 2011 195 (5): 709–720. doi: https://doi.org/10.1083/jcb.201102131

Hematopoietic stem cells (HSCs) give rise to all lineages of blood cells. Because HSCs must persist for a lifetime, the balance between their proliferation and quiescence is carefully regulated to ensure blood homeostasis while limiting cellular damage. Cell cycle regulation therefore plays a critical role in controlling HSC function during both fetal life and in the adult. The cell cycle activity of HSCs is carefully modulated by a complex interplay between cell-intrinsic mechanisms and cell-extrinsic factors produced by the microenvironment. This fine-tuned regulatory network may become altered with age, leading to aberrant HSC cell cycle regulation, degraded HSC function, and hematological malignancy.

Introduction

Hematopoiesis is the lifelong process by which all the cells of the blood system are produced in a hierarchical manner from a small population of hematopoietic stem cells (HSCs), which reside in the bone marrow (BM) cavity in adult mammals (Orkin and Zon 2008). HSCs give rise to progenitor cells that become increasingly lineage restricted and ultimately differentiate into all lineages of mature blood cells. As HSCs continually replenish cells that are lost or turned over, they must self-renew to maintain themselves over the lifetime of the organism. HSC self-renewal is experimentally defined as the capacity for long-term reconstitution of all blood lineages upon transplantation into a recipient (Ema et al., 2006). However, the capacity to self-renew is by itself insufficient for lifelong maintenance of a functional HSC compartment, as the accumulation of damage in such long-lived cells can result in dysfunctional hematopoiesis including BM failure or leukemic transformation (Lane and Gilliland 2010). Adult HSCs reside in specialized microenvironments, known collectively as the BM niche (Schofield 1978 Wilson and Trumpp 2006), where they are maintained in a quiescent, or dormant, state. It is believed that quiescence contributes to HSC longevity and function, perhaps in part by minimizing stresses due to cellular respiration and genome replication (Eliasson and Jönsson 2010).

In this review, we will focus on mouse hematopoiesis and explore the balance between HSC quiescence and proliferation, and how these two processes are regulated by intrinsic and extrinsic factors. We will also address the effects of aging on the mechanisms of HSC proliferation and quiescence, and the consequences of aging on HSC function and leukemic transformation.

Developmental origin of HSCs

Although HSCs reside in the BM in adults, this is merely the endpoint of an otherwise nomadic journey during embryogenesis. Moreover, the quiescent state of HSCs in the adult BM is reached only after a period of active cell cycling and proliferation to generate the blood system during fetal life (Bowie et al., 2006). Hematopoiesis in the embryo is considered to occur in successive waves, with the initial “primitive” wave geared toward the rapid production of red blood cells for oxygen transport but with little HSC activity the second, or “definitive” wave, is characterized by the generation of all lineages of blood cells and the production of the first engrafting HSCs. Primitive hematopoiesis occurs as early as day E7.5 in the yolk sac blood islands (Palis et al., 1999 Medvinsky et al., 2011). The definitive wave of hematopoiesis, on the other hand, occurs in parallel in several tissues over a more protracted period of time. Definitive HSCs are found in the aorta-gonad-mesonephros (AGM) region and the placenta by E8.5 and E10, respectively, as well as in the yolk sac (Medvinsky and Dzierzak 1996 Gekas et al., 2005 Samokhvalov et al., 2007). Subsequently, HSCs from one or more of these sites expand in the fetal liver during the remainder of embryonic life, while their production by the AGM and placenta become extinguished (Medvinsky et al., 2011).

By E17.5 and through the first two weeks of postnatal life, HSCs leave the liver to colonize the bones via an active recruitment mechanism involving the CXCL12/SDF-1 chemokine receptor CXCR4 (Ma et al., 1998), which regulates HSC homing and engraftment in the nascent BM environment by activating the guanine nucleotide exchange factor Vav1, which in turn regulates the GTPases Rac and Cdc42 (Cancelas et al., 2005 Sanchez-Aguilera et al., 2011). Other factors also contribute to HSC localization to the BM either in conjunction with CXCR4, such as prostaglandin E2 (PGE2) and the neuronal guidance protein Robo4 (Hoggatt et al., 2009 Smith-Berdan et al., 2011), or independently from CXCR4 like c-Kit, the calcium-sensing receptor (CaR), and the transcription factor Egr1 (Christensen et al., 2004 Adams et al., 2006 Min et al., 2008). Thereafter, HSCs remain anchored in the BM niche by complex integrin-dependent mechanisms (Scott et al., 2003 Forsberg and Smith-Berdan 2009), though small numbers of HSCs will periodically migrate from the BM into the circulation and back for short periods of time under homeostatic conditions, perhaps as a form of immunosurveillance (Massberg et al., 2007 Bhattacharya et al., 2009). Taken together, these data underscore the dynamic nature of hematopoietic development from embryogenesis through adulthood.

Distinct cell cycle activities in fetal and adult HSCs

The cell cycle activity of HSCs over the lifetime of an organism is equally dynamic, and reflects the needs of the organism at different developmental stages. During fetal life, the central function of HSCs is to rapidly generate homeostatic levels of blood cells for oxygen transport and immune system development in the growing organism. In line with this role, between 95 and 100% of HSCs are actively cycling in the mouse fetal liver with a cell cycle transit time between 10–14 h (Fig. 1 Bowie et al., 2006 Nygren et al., 2006).

Although HSC residence in the BM during adulthood is often associated with quiescence, HSCs do not appear to become quiescent immediately upon seeding the BM, as all HSC activity remains confined to the fraction of actively cycling lineage-negative (Lin − ) BM cells in 3-wk-old weanling mice (Bowie et al., 2006). Remarkably, the BM HSC population rapidly switches to a quiescent state by 4 wk of age, with only ∼5% of total HSCs actively in the cell cycle (defined as S, G2, or M phases) thereafter through adult life (Cheshier et al., 1999 Bowie et al., 2006 Kiel et al., 2007) (Fig. 1). This abrupt change in HSC proliferation activity suggests that HSC quiescence is not solely linked to their localization in the BM cavity, but may reflect feedback mechanisms informing HSCs that blood cell formation has reached homeostatic levels, or that the development of the BM niche has been completed. Transition from active cell cycling in fetal HSCs to quiescence in adult HSCs is also associated with changes in gene expression programs, including a marked reduction in expression of Sox17, a transcription factor required for the maintenance of fetal but not adult hematopoiesis (Kim et al., 2007).

Interestingly, adult HSCs are not uniformly dormant. In vivo experiments assessing cell cycle activity (by measuring retention of BrdU or histone 2B (H2B)-GFP expression pulses) in mature HSCs suggest a notable heterogeneity in the degree to which HSCs are quiescent (Wilson et al., 2008 Foudi et al., 2009). These studies propose the subfractionation of the HSC compartment into “dormant” and “activated” phenotypes with distinct rates of cell cycle entry, comprising ∼5–10% and 90–95% of the HSC pool, respectively (Fig. 1). Dormant HSCs are computed to divide only once every 145 d or more, and appear to be enriched for long-term reconstitution potential. This small population of cells may represent a reservoir of HSC activity kept aside in the adult BM to be called upon only by severe hematopoietic injury, thus ensuring the maintenance of blood homeostasis. However, recent work in human blood cells indicates that human HSCs enter the cell cycle on average once every 40 wk (Catlin et al., 2011). Although this finding underscores the considerable physiological difference between humans and rodents that must be kept in mind when interpreting studies performed in the mouse, it also appears to support the hypothesis that limiting cell cycle activity is critical to lifelong HSC maintenance.

Despite the great difference in the frequency by which fetal and adult HSCs divide, once in the cell cycle, they transit through it at the same slow rates compared with their more differentiated progenitor cells due to an extended passage through the G1 phase of the cell cycle (Nygren et al., 2006). Thus, the decision regarding whether HSCs enter the cell cycle, as opposed to how they progress through it, appears to be one of the essential differences between fetal and adult hematopoiesis. Moreover, disruption of HSC quiescence leads to defects in HSC self-renewal and often results in HSC exhaustion (Orford and Scadden 2008), hence underscoring the critical importance of a constitutively low level of cell cycle activity for proper function of the blood system during adult life.

Together, these findings underscore the requirement for a complex network of regulatory mechanisms in enforcing the balance between HSC quiescence and proliferation, and the proper maintenance of blood homeostasis in the adult organism. In the following sections, we will discuss the current understanding of such intrinsic and extrinsic regulatory mechanisms in adult HSCs and highlight the relevant genetic mouse models that have contributed to this understanding (Table I).

Cell-intrinsic mechanisms regulating HSC quiescence

Numerous factors regulate the cell cycle status within a cell however, they can largely be reduced to the competing actions of Cdks, which drive cell cycle progression, and Cdk inhibitors (CKIs), which blunt progression through the cell cycle (Morgan 1997). A large body of studies has begun to unravel the molecular wiring within adult HSCs that mediate their continued maintenance in a quiescent, or G0, state, while allowing for their rapid entry into the cell cycle to respond to hematopoietic demand (Fig. 2).

Rb Family.

The retinoblastoma (Rb) family of transcriptional repressors, including the pRb, p107, and p130 proteins, restricts cell cycle entry by repressing E2F gene transcription of positive cell cycle regulators, which include E-type cyclins. In their hypophosphorylated state, Rb family members restrict entry through G1 phase. However, upon phosphorylation by the cyclin D–Cdk4/6 complex, Rb family members are partially inactivated and permit cell cycle progression through G1. Subsequent phosphorylation by cyclin E–Cdk2 further inactivates Rb-mediated inhibition of E2F, resulting in G1 exit and entry into S phase. A firm role for Rb family members in the control of HSC quiescence was not established until recently, as considerable functional redundancy exists within this family and as all members are expressed, albeit at different levels, in HSCs (Passegué et al., 2005). For instance, no hematopoietic phenotype was observed in p130-deficient mice (Cobrinik et al., 1996), and p107 deletion resulted in only a mild myeloid hyperplasia (LeCouter et al., 1998). Removal of pRb had also no effect on HSC self-renewal as assessed by serial transplantation (Walkley and Orkin 2006), and although pRb-deficient mice exhibited myeloid expansion, this was shown to be a non-cell autonomous effect (Walkley and Orkin 2006 Walkley et al., 2007). Strikingly, however, conditional deletion of all three Rb family members in adult mice resulted in a robust cell-intrinsic myeloproliferation phenotype leading to the death of the animals by 1–3 mo after gene inactivation (Viatour et al., 2008). This was accompanied by an increase in both HSC proliferation and absolute cell numbers, and by severe defects in HSC self-renewal as BM from mice deficient in all three Rb family genes had grossly impaired reconstitution after transplantation (Viatour et al., 2008). Taken together, these findings indicate that Rb family members play critical, albeit overlapping roles in the regulation of HSC quiescence and continued self-renewal activity.

D-cyclins and Cdk4/6.

Cyclins and Cdks act upstream of the Rb family members to mediate cell cycle entry and progression. As the regulation of HSC quiescence fundamentally involves controlling whether HSCs enter the G1 phase of the cell cycle or remain in G0, the activity of the cyclin D–Cdk4/6 complex, which controls progression through G1 in response to mitogenic signals, is likely a central determinant of HSC cell cycle activity. The D-cyclin family includes cyclin D1 (Ccnd1), cyclin D2 (Ccnd2), and cyclin D3 (Ccnd3), which are all expressed, albeit at different levels, in HSCs (Passegué et al., 2005). Similar to the Rb gene family, mice deficient for a single D-cyclin, or for only one of the two associated Cdks, have minimal hematopoietic defects, hence illustrating the considerable functional redundancy protecting this complex (Fantl et al., 1995 Malumbres et al., 2004). However, mice deficient in all three D-cyclins die during late embryogenesis due to heart defects and hematopoietic failure with significant reduction in peripheral red blood cell numbers (Kozar et al., 2004). Furthermore, D1/2/3-cyclins −/− mice have lower numbers of HSCs and progenitor populations in the fetal liver, with decreased frequency of HSCs in S and G2-M stages of the cell cycle. Fetal liver cells derived from D1/2/3-cyclins −/− mice are also unable to provide short-term reconstitution of irradiated recipient mice (Kozar et al., 2004). Cdk4/6 −/− mice also display late embryonic lethality accompanied by a defect in fetal hematopoiesis very similar to the phenotypes observed in D1/2/3-cyclins −/− mice, including severely decreased numbers of proliferating erythroid progenitors in the fetal liver and of red blood cells circulating in the peripheral blood (Malumbres et al., 2004). These findings further underscore the essential requirement for active HSC proliferation during fetal blood development and stress the importance of the cyclin D–Cdk4/6 complex in fetal HSC cell cycle progression. However, it is yet to be established whether there is a different requirement for the cyclin D–Cdk4/6 complex in maintaining the proliferation and functionality of adult HSCs.

Ink4 family.

The Ink4 family includes the CKIs p15 Ink4b , p16 Ink4a , p18 Ink4c , and p19 Ink4d , and a functionally distinct protein, p19 ARF that is encoded by an alternate reading frame within the Ink4a locus, which also encodes p16 Ink4a (Sherr 2001). The Ink4 proteins all function as antagonists of the cyclin D–Cdk4/6 complex, thereby blocking phosphorylation of Rb family members and subsequent entry into S phase. p19 ARF , on the other hand, is not a CKI and instead positively regulates p53 (see CIP/KIP family section Sherr 2001). Deletion of both p16 Ink4a and p19 ARF through the disruption of the entire Ink4a locus has minimal consequences for HSC activity, consistent with the reported low or absent expression of these factors in HSCs (Passegué et al., 2005). Their limited expression is likely the result of transcriptional repression by Bmi1, a polycomb group family member and chromatin remodeler that is expressed preferentially in HSCs and actively represses the Ink4a locus (Lessard and Sauvageau 2003 Park et al., 2003). Bmi1 deficiency is lethal in adult mice due to hematopoietic failure caused by a progressive depletion of HSCs (Lessard and Sauvageau 2003 Park et al., 2003). This result implies that the resulting increase in p16 Ink4a and p19 ARF expression caused by Bmi1 removal may completely inhibit the infrequent cell cycle entry of adult HSCs, which is essential for HSC self-renewal and maintenance of blood homeostasis. The fact that combined deletion of p16 Ink4a and p19 ARF rescues the majority of the hematopoietic phenotypes in Bmi1-deficient mice supports this notion and indicates a critical role for Bmi1 in restraining p16 Ink4a and p19 ARF expression in adult HSCs (Oguro et al., 2006). In contrast, deletion of p18 Ink4c directly results in increased numbers of actively cycling HSCs without impairment of HSC self-renewal activity, as p18 Ink4c -deficient cells confer enhanced reconstitution to lethally irradiated recipients (Yuan et al., 2004). Notably, p18 Ink4c expression is highest in quiescent HSCs, and is significantly decreased in actively cycling fetal HSCs relative to adult HSCs, consistent with its role as a brake for HSC proliferation (Passegué et al., 2005 Bowie et al., 2007). This result may also provide some molecular basis for the difference in cell cycle activity between fetal and adult HSCs. Collectively, these data indicate that several of the Ink4 family members are differentially regulated in adult HSCs to maintain the proper balance between quiescence and proliferation, with p18 Ink4c directly acting to restrict cell cycle entry, and Bmi1 preventing expression of p16 Ink4a and p19 ARF .

CIP/KIP family.

The CIP/KIP family includes the CKIs p21 Cip , p27 Kip1 , and p57 Kip2 , which also restrain entry into S phase by inhibiting the activity of the cyclin E–Cdk2 complex. p21 Cip is expressed at somewhat greater levels in adult HSCs relative to their differentiated progeny or to fetal HSCs (Passegué et al., 2005 Bowie et al., 2007). Although a role for p21 Cip in regulating HSC quiescence had initially been suggested (Cheng et al., 2000a), more recent reports using different mouse backgrounds and/or methodologies suggest that the function of p21 Cip in regulating HSC cell cycle activity may be restricted to periods of stress rather than during homeostasis (Cheng et al., 2000a van Os et al., 2007 Foudi et al., 2009). Aside from p21 Cip , analysis of p27 Kip1 -deficient mice suggests that p27 Kip1 deficiency alone has a limited impact on HSC function and instead appears to affect the cell cycle activity of more committed progenitor populations (Cheng et al., 2000b). Interestingly, although earlier studies detected no overt hematopoietic defects in p57 Kip2 -deficient mice (Yan et al., 1997 Zhang et al., 1997), more recent work using hematopoietic-specific deletion of p57 Kip2 in adult mice suggests that p57 Kip2 is in fact critical for maintaining HSC quiescence (Matsumoto et al., 2011). In addition, the defective quiescence phenotype of p57 Kip2 -deficient HSCs is exacerbated by concomitant deletion of either p21 Cip or p27 Kip1 , and p27 Kip1 overexpression is able to compensate for the loss of p57 Kip2 (Matsumoto et al., 2011 Zou et al., 2011). Taken together, these studies reveal p57 Kip2 as a critical regulator of HSC quiescence in adult mice, and suggest some degree of functional redundancies among the CIP/KIP family of CKIs. Interestingly, growth-repressive signals such as TGF-β directly target the p21 Cip and p57 Kip2 genes, suggesting a role for the BM niche in regulating expression of these CKIs in HSCs (Scandura et al., 2004). Interestingly, p53, a master transcriptional regulator that induces the transcription of p21 CIP in addition to a plethora of other genes upon cellular insult, also regulates HSC quiescence (Asai et al., 2011). p53 −/− HSCs have increased BrdU incorporation and decreased frequency of G0 cells (Liu et al., 2009). Although p53 deletion increases the numbers of phenotypic HSCs, transplantation experiments demonstrate that p53 −/− HSCs actually have decreased functionality, suggesting that p53 positively regulates self-renewal, perhaps by restraining HSC activity (TeKippe et al., 2003 J. Chen et al., 2008). However, this appears to be independent of p21 Cip , and may instead be due to the transcription of other p53 targets that negatively regulate HSC proliferation, particularly Necdin and Gfi-1 (Liu et al., 2009).

PI3K signaling pathway.

The phosphatidylinositol-3 kinase (PI3K) signaling pathway integrates numerous upstream signals from activated cytokine receptors and other mitogenic stimuli to drive cell proliferation, growth, and survival. Downstream targets of the PI3K pathway include the threonine/serine kinase Akt, which can both lead to the activation of mammalian target of rapamycin (mTOR) and the suppression of the forkhead box O (FoxO) family of transcription factors. This pathway is restrained through the action of the tumor suppressor phosphatase and tensin homologue (PTEN). Specifically, PI3K signaling may accelerate cell proliferation by stabilizing D-type cyclins and inhibiting FoxO-dependent transcription of p21 Cip and p27 Kip1 (Massagué 2004). Attenuation of signaling through the PI3K pathway is essential to preserve HSC quiescence and long-term self-renewal (Warr et al., 2011). Conditional deletion of Pten in the hematopoietic system results in an aggressive and deadly early-onset myeloproliferative neoplasm (MPN), which is accompanied by a threefold increase in the frequency of cycling HSCs (Yilmaz et al., 2006 Zhang et al., 2006). Pten −/− HSCs also show defective self-renewal activity as they are unable to provide long-term engraftment in transplanted mice, which results in the depletion of the HSC pool in Pten-deficient mice (Yilmaz et al., 2006 Zhang et al., 2006). Increased HSC cycling and defective self-renewal activity are also observed in a myristoylated Akt retroviral transplantation model, which has constitutive Akt activity (Kharas and Gritsman 2010), and in mice lacking the tumor suppressor Tsc1, which has unchecked mTOR activity (C. Chen et al., 2008). Furthermore, rapamycin, a pharmacological inhibitor of mTOR, is able to reverse many of the phenotypes associated with Pten deficiency, including the increased proliferation of HSCs (Yilmaz et al., 2006). These results suggest that PTEN normally restrains HSC entry into the cell cycle entry, at least in part, through inhibition of downstream mTOR signaling. However, as discussed previously, constitutive levels of Akt also repress the activity of FoxO family members. Interestingly, FoxO3 −/− and FoxO1/3/4 −/− mice display increased HSC cell cycle activity accompanied by increased levels of reactive oxygen species (ROS), which can be reversed after the administration of the ROS scavenger N-acetylcysteine (NAC Miyamoto et al., 2007 Tothova et al., 2007). In contrast, loss of PI3K signaling caused by deletion of Akt1/2 results in an increased proportion of HSCs in G0/G1 phase and decreased HSC proliferation, which can be rescued in vitro by pharmacologically increasing cellular ROS levels (Juntilla et al., 2010). Although cellular oxygen levels and HSC proliferation are thought to be directly associated (Ito et al., 2006 Eliasson and Jönsson 2010 Takubo et al., 2010), a direct demonstration of the function of ROS in controlling HSC cell cycle in vivo remains to be provided. Collectively, these studies suggest that HSC quiescence is preserved at least in part by limiting activation of the PI3K signaling pathway, which may maintain high levels of FoxO-dependent expression of CKIs and minimize ROS production, thereby blocking HSC cell cycle progression.

Cell-extrinsic mechanisms regulating HSC quiescence

Although quiescence is essential for the self-renewal of adult HSCs, they must nonetheless retain the capacity to proliferate rapidly, albeit transiently, in response to extrinsic cues that signal injury or infection. Evidence that cell-extrinsic signals regulate the cell-intrinsic mechanisms governing HSC quiescence can be inferred from the observation that HSCs enter the cell cycle upon pharmacological mobilization in vivo or culture ex vivo (Passegué et al., 2005), and from experimental evidence indicating that osteoblasts and other cellular components of the BM niche influence HSC cell cycle status (Wilson et al., 2007).

Part of this mechanism proceeds via a series of conserved developmental pathways, including TGF-β, Wnts, Notch ligands, and the Hedgehog (Hh) pathway, which are all essential for embryonic development and fetal hematopoiesis. Deletion of TGF-β1 or Indian hedgehog (Ihh) lead to defects in fetal erythropoiesis resulting in embryonic lethality (Dickson et al., 1995 Cridland et al., 2009), whereas Wnt3a-deficient mice show depressed numbers of HSCs in the fetal liver (Luis et al., 2009). However, conclusive in vivo evidence for their role in adult HSC cell cycle regulation has been difficult to generate, as Mx-Cre–driven conditional deletion of Notch, Wnt, and Hh signaling components in adult mice do not reveal clear hematopoietic defects or alterations in HSC cell cycle activity (Koch et al., 2008 Maillard et al., 2008 Gao et al., 2009). This suggests that they are either not absolutely critical in the context of adult hematopoiesis, or that significant functional redundancy exists between these and other niche signals. Additional work will be required to disentangle the complex direct and indirect effects of these developmental pathways on adult HSCs in vivo, as well as the relevant receptor–ligand interactions on both HSCs and/or BM niche cells that mediate them. Furthermore, it should be emphasized that these pathways may also act as critical regulators of HSC cell cycle activity in specific hematological contexts that have not yet been well studied, such as stress or disease (Zhao et al., 2007, 2009)

A wide range of environmental factors, including cytokines, growth factors, and other mediators produced by circulating immune cells and cells comprising the BM niche, also modulate HSC quiescence. In contrast to the previously discussed developmental pathways, these environmental factors, which include the chemokine CXCL12/SDF-1, stem cell factor (SCF), angiopoietin-1 (Ang-1), and thrombopoietin (TPO), are dispensable for fetal hematopoiesis, but are essential to regulate HSC quiescence in adult organisms (Wilson et al., 2009). Mice deficient in Cxcr4 (SDF-1 receptor), Tpo, Tie-2 (Ang-1 receptor), or carrying a hypomorphic allele of Kit (SCF receptor), all exhibit normal fetal hematopoiesis followed by increased cell cycle activity and progressive loss of the HSC compartment during adult life (Nagasawa et al., 1996 Puri and Bernstein 2003 Arai et al., 2004 Qian et al., 2007 Nie et al., 2008 Thorén et al., 2008). Collectively, these results highlight the importance of cell-extrinsic mechanisms in regulating HSC function, and further underscore the importance of quiescence for the maintenance of the HSC compartment during adulthood.

A body of evidence suggests that the regulation of lipid raft clustering on the surface of HSCs may be a critical determinant of HSC quiescence by dictating the level of Akt activation induced by cytokine receptors. Quiescent HSCs show minimal amounts of lipid raft clustering, while actively proliferating hematopoietic progenitor cells have high levels of clustering and Akt pathway activation (Yamazaki et al., 2006, 2007). In addition, SCF or TPO stimulation of HSCs in in vitro culture conditions lead to lipid raft clustering, rapid Akt phosphorylation, and exclusion of FoxO3a from the nucleus, resulting in decreased expression of p21 Cip and p57 Kip2 and reentry of HSCs into the cell cycle (Yamazaki et al., 2006, 2007). Interestingly, TGF-β signaling suppresses lipid raft clustering in HSCs, hence limiting the activation of Akt induced by SCF or TPO stimulation to a level that likely promotes HSC survival but not proliferation (Yamazaki et al., 2009). TGF-β also directly induces expression of p57 Kip2 , and prevents sequestration of cyclin D1 (Yamazaki et al., 2009 Scandura et al., 2004). Taken together, these results explain a mechanism by which TGF-β may enforce HSC quiescence, although the relationship between TGF-β and HSC quiescence has not been directly addressed in vivo due to the confounding effect of an autoimmune condition present in mice lacking TGF-β signaling components (Larsson et al., 2003). These data may also help explain why SCF and TPO are associated with HSC quiescence and not proliferation in vivo, as factors such as TGF-β that are present in the BM niche may prevent activation of Akt by these cytokines. Furthermore, TGF-β also collaborates with Notch ligands to induce expression of p21 Cip via a mechanism involving the transcription factors junB and Hes1 (Yu et al., 2006 Santaguida et al., 2009). Mice deficient in the transcription factor Smad4, which interacts with TGF-βR–activated Smad2/3, express lower levels of Notch1 but the effect of Smad4 deficiency on HSC cell cycle activity has not yet been directly studied (Karlsson et al., 2007). These findings suggest that TGF-β and Notch may function in an interdependent manner to regulate HSC cell cycle activity. In addition, the induction of p21 Cip and p57 Kip2 expression is also regulated by a number of other BM niche-associated factors, including TPO, Wnt, and SDF-1 (Qian et al., 2007 Fleming et al., 2008 Nie et al., 2008). Collectively, these data indicate that multiple signaling mechanisms activated by developmental pathways and environmental factors may converge upon the regulation of CKI expression, which in turn might contribute to the prolonged passage of HSCs through the G1 phase of the cell cycle.

Regulation of cyclin expression forms another convergence point by which extrinsic signals regulate the internal machinery governing quiescence in adult HSCs. Mice deficient in Cxcr4 have increased levels of cyclin D1, which suggests that the CXCR4–SDF-1 pathway impacts on the activity of the cyclin D–Cdk4/6 complex and G1 phase progression in HSCs (Nie et al., 2008). Notably, TGF-β and Notch ligands up-regulate CXCR4, and this may account for some of their quiescence-enforcing activity (Franitza et al., 2002). On the other hand, activation of the Wnt and Hh pathways is associated with increased expression of cyclin D1 in HSCs, although Wnt signaling is typically associated with maintenance of HSC quiescence and self-renewal activity (Fleming et al., 2008 Merchant et al., 2010). It may be that signaling through these pathways helps to maintain basal expression of cyclin D1 in quiescent HSCs (Passegué et al., 2005). Such careful balancing of pro- and anti-proliferative factors in the BM niche could account for their role in enforcing a homeostatic level of HSC quiescence, while leaving HSCs sufficiently primed for proliferation in response to hematopoietic demand. Recent work suggests that interferons, a family of inflammatory cytokines, rapidly induce HSC cell cycle entry, and this may occur at least in part via activation of the Akt pathway (Essers et al., 2009 Baldridge et al., 2010). These results confirm that the quiescent state of HSCs is indeed quickly reversible, which is likely to be critical for effective control of stress conditions such as infection or blood loss.

Taken together, these studies demonstrate that quiescence is a dynamic condition that is orchestrated by a careful balance between collaborating and opposing extrinsic signals acting on various components of the intrinsic cell cycle machinery, which directly control HSC proliferation and self-renewal activity (Fig. 2). Further studies of the mechanisms by which these extrinsic factors regulate the proliferation of fetal vs. adult HSCs will be invaluable in understanding the regulation of the cell cycle machinery in HSCs and other stem cells as organisms reach maturity. A careful analysis of how these extrinsic factors may contribute to the heterogeneity observed in the cycling activity of HSC subpopulations should also prove highly informative (Challen et al., 2010).

Aging and the control of HSC cell cycle activity

The hematopoietic system declines with age, resulting in a diminished production of adaptive immune cells, termed immunosenescence, and an increased incidence of anemia and myeloid malignancies (Fig. 1 Beerman et al., 2010b). Although aged mice have increased numbers of phenotypic HSCs, the functional capacity of these cells is actually compromised, as evidenced by the decrease in long-term multilineage reconstitution potential of purified old HSCs transplanted into young recipient mice (Rossi et al., 2005). However, whether this is a result of a true change in self-renewal versus a clonal change in the composition of the HSC compartment is currently debated (Cho et al., 2008 Waterstrat and Van Zant 2009 Beerman et al., 2010a).

During aging, the additive effects of repeated cellular and genomic insults can become apparent, potentially damaging the integrity of the HSC pool (Blanpain et al., 2011). Indeed, mice deficient for key DNA repair pathway components exhibit diminished HSC self-renewal with age and early functional exhaustion (Nijnik et al., 2007 Rossi et al., 2007). In fact, long-lived quiescent HSCs may be particularly susceptible to the accumulation of DNA damage over time, as their cell cycle status forces them to repair DNA damage using the error-prone and mutagenic nonhomologous end joining (NHEJ) mechanism (Mohrin et al., 2010). Moreover, HSCs from aged mice exhibit increased phosphorylated γ-H2AX staining, which indicates increased occurrence of DNA double-strand breaks, and suggests that aging may contribute to the loss of HSC function in part due to genomic instability (Rossi et al., 2007). In addition to DNA damage, the short-circuiting or overload of epigenetic control mechanisms regulating HSC self-renewal may also contribute to the pathogenesis of hematological malignancies, particularly in the myeloid lineage, later in life (Rossi et al., 2008). Indeed, deletion of Ten-Eleven-Translocation-2 (Tet2), a factor thought to regulate gene silencing via methylation and which is commonly mutated in individuals with MPNs, leads to aberrant activation of myeloid differentiation gene programs in HSCs and subsequent MPN emergence in mice (Ko et al., 2011, Li et al., 2011, Moran-Crusio et al., 2011, Quivoron et al., 2011). Thus, the appropriate control of HSC cell cycle activity may therefore become particularly important in an aging organism as a means of preventing the outgrowth of damaged clones via induction of cell cycle arrest or cellular senescence.

Studies of aged mice show an overall decrease in HSC cell cycle activity, with old HSCs undergoing fewer cell divisions than young HSCs as assessed by BrdU and N-hydroxysuccinimidyl biotin (NHS-biotin) incorporation (Janzen et al., 2006 Nygren and Bryder 2008). These results suggest that Cdks and other activators of the cell cycle may undergo functional decline or that the activity of certain cell cycle checkpoint mechanisms such as CKIs may increase with age, hence delaying HSC entry into the cell cycle. This latest hypothesis is supported by the observations of increased p16 INK4a levels in old HSCs and of better engraftment capacity and increased cell cycle activity in old p16 INK4a−/− HSCs compared with old wild-type HSCs (Janzen et al., 2006). As increased expression of p16 Ink4a is associated with aging in numerous tissues, and its expression correlates with cellular senescence (Sharpless and DePinho 2007), it is appealing to suggest that p16 Ink4a expression in aged HSCs may result in the cellular senescence of at least a subpopulation of old HSCs. However, another group demonstrates no correlation between p16 Ink4a expression levels and HSC aging (Attema et al., 2009). Such discrepancies may in part be due to heterogeneity among cohorts of aged mice, or the sensitivity of different methods used to detect p16 Ink4a expression. Interestingly, Bondar and Medzhitov (2010) elegantly demonstrated that cell competition exists in the HSC compartment and is dependent on the relative levels of p53 expression, and a balance between proliferation and senescence responses. This work shows that cells with lower levels of p53 outcompete cells with higher p53 levels for BM engraftment, resulting in growth arrest and the induction of a senescence-related gene expression program in the outcompeted cells, which includes p16 Ink4a expression. Furthermore, HSCs from old p53 +/m mice, which carry a hypermorphic allele of p53, exhibit decreased proliferation and self-renewal capacity relative to old p53 +/+ controls, whereas old p53 +/− HSCs, which have only one functional p53 allele, exhibit increased function compared with controls (Dumble et al., 2007). Notably, such differences are not observed in HSCs from young mice, regardless of p53 status. Collectively, these data suggest that p53-dependent checkpoint mechanisms, perhaps including but not limited to induction of p16 Ink4a , may in part underlie the functional decline and decreased cell cycle activity of aged HSCs. This might be due to a combination of cellular senescence in some HSCs amid an expansion of other functionally compromised HSC clones in which cell cycle checkpoints have been compromised. Such a model may help explain the apparent lack of senescent HSCs observed in the expanded, but functionally impaired, HSC compartment of old mice, while at the same time accounting for the improved function of the HSC pool in aged p16 INK4a−/− mice, where otherwise competitively disadvantaged HSC populations may still contribute to blood production.

As described previously, mouse models that lead to unchecked levels of mTOR activity result in a deregulated HSC compartment and stem cell exhaustion (Yilmaz et al., 2006 Zhang et al., 2006 C. Chen et al., 2008). Recent work also indicates that mTOR activity is increased in aged HSCs (Chen et al., 2009). Interestingly, pretreatment of aged mice with rapamycin, which inhibits mTOR activity, rescues many of the functional defects observed in old HSCs, including their decreased cell cycle activity and diminished engraftment (Chen et al., 2009). At the moment it is quite challenging to integrate this observation with the aforementioned increase in p16 Ink4a expression exhibited by the old HSCs because increased mTOR activity results in increased HSC cycling, at least in young Pten −/− and Tsc1 −/− HSCs (Yilmaz et al., 2006 C. Chen et al., 2008). However, it is plausible that in aged mice, mTOR levels do not reach a similar threshold as observed in these mutant cells, or that mTOR signaling exerts differential effects in dysfunctional old HSCs.

Conclusions and future perspectives

Hematopoiesis is a dynamic process that has evolved to generate and maintain homeostatic levels of blood cells for the lifetime of the organism. The lifelong persistence of HSCs is likely due to their acquisition of a quiescent phenotype upon maturity of the host, as well as their localization to specialized niches in the BM cavity wherein extrinsic cues regulate their cell cycle activity. Both of these features can be envisioned as strategies to limit the frequency and severity of potentially damaging replicative stresses. The aging hematopoietic system serves as a compelling model in which to examine the limitations of these protective strategies. The work reviewed here suggests that an old hematopoietic system, in which HSCs may have acquired damage, must deal with the conflicting requirements of suppressing the outgrowth of damaged and/or transformed clones, while at the same time producing enough blood cells to maintain homeostasis. The biological response to this problem appears to err on the side of caution, as evidenced by the decreased self-renewal potential and reduced cell cycle entry observed in the expanded pool of old HSCs. Although it would be tempting to explain these responses as a p53- and/or p16 Ink4a -driven program of cellular senescence that could underlie some of the hematological insufficiencies observed in the elderly, experimental evidence is still needed to confirm this hypothesis. The high incidence of hematological malignancy observed with age suggests that the ability to arrest damaged HSCs is likely not without fundamental limitations, hence supporting the notion that expansion of functionally deficient HSC clones harboring defective cell cycle checkpoints contributes to the aging of the blood system (Kastan and Bartek 2004 Bondar and Medzhitov 2010).

Considerable work still remains to be done to fully understand how components of the regulatory networks that control cell cycle activity in young adult HSCs may become altered with age. For instance, is there a global change in the expression levels of master cell cycle regulators in old HSCs? Although microarray analyses have been performed, they do not uncover any particular changes in cell cycle machinery or upstream regulatory genes in old HSCs, though alterations in the expression of genes involved in myeloid and lymphoid differentiation and epigenetic regulation are observed (Rossi et al., 2005 Chambers et al., 2007). These results suggest that the activity, rather than the expression level, of cell cycle regulators is altered in old HSCs, which is consistent with the work showing increased mTOR activity in old HSCs (Chen et al., 2009). Furthermore, as the activation level of the cell cycle machinery is in part regulated by cell-extrinsic signals, it will be critical to understand the extent to which the extracellular environment where HSCs reside, both in the niche and systemically, is altered with age and contributes to the functional decline of the hematopoietic system.

Taken together, these findings underscore the challenges inherent in understanding the cell cycle regulation of a system as complex and dynamic as the aging hematopoietic system, as aging itself appears to alter the cellular context in which these regulatory mechanisms operate. It can be speculated that the functional decline of the blood system could be in part due to a clonal expansion of damaged HSC populations that outcompete HSCs with intact cell cycle checkpoints. Thus, experimental approaches that uncover the extent to which distinct subpopulations of HSCs and their output change over time will likely be crucial to our understanding of how the phenotypic features of aged hematopoiesis arise. Understanding how aging affects HSC biology will also provide critical insight into the pathogenesis of hematological malignancies that result from such an outgrowth of damaged HSCs. In turn, the development of targeted therapies that modulate the activity of cell cycle checkpoints in HSCs may prove efficacious in eliminating damaged HSC clones or in reducing their output. Such therapies may reestablish favorable conditions for the activation of undamaged HSCs and a restoration of effective hematopoiesis in aged individuals or patients with hematological malignancies. Moreover, the results of studies using HSCs as a developmental model system may be broadly applicable to other tissues in which stem cells are either not as well characterized or as easily isolated. Thus, the continued elucidation of the mechanisms controlling cell cycle activity in fetal, adult, and old HSCs will have significant impact at the intersection between cancer and stem cell biology, both at the bench and in the clinic.


Introduction

Host immunity requires a constant renewal of red blood cells and leukocytes throughout life, as these cells have a restricted life span. Hematopoietic cell turnover is enhanced following acute stress situations, such as infections or irradiation, by the proliferation of hematopoietic stem cells (HSCs) and progenitor cells (HPCs), which respond to these conditions. The hematopoietic stem and progenitor cells (HSPCs) are a small population of undifferentiated cells that reside in the bone marrow (BM). HSCs are defined by their capacity for self-renewal and ability to differentiate into all blood cell lineages. Another distinct feature of these cells is their ability to migrate out of the BM to the peripheral blood. This process is enhanced under stress as a part of the host mechanisms of defense and repair. In addition, HSCs injected to the blood stream, as performed in BM transplantation, can home to the BM and reestablish the HSC pool as a lifelong reservoir of new blood and immune cells [1].

The BM is the main site of adult hematopoiesis, and the majority of HSPCs remain confined to the BM microenvironment in a quiescent nonmotile state maintained via adhesive interactions [2–4]. Under stress conditions, undifferentiated progenitor cells can be triggered by their microenvironment to undergo enhanced proliferation and differentiation, to address the demand of the immune and hematopoietic systems for new leukocytes and blood cells.

Cells in the BM microenvironment maintain a functioning pool of precursor cells regulated by cytokines, by chemokines, and by additional lipid effectors. The chemokine CXCL12 and its primary receptor CXCR4 are essential for adhesion and retention of HSPCs in the mouse BM [5,6]. During homeostasis in the steady-state, CXCR4 is expressed by hematopoietic cells in addition to stromal cells, which are the main source for CXCL12 in the BM. CXCR4 + HSPCs tightly adhere to BM stromal cells, which express functional, membrane-bound CXCL12 [6]. The CXCL12/CXCR4 pathway is involved in regulation of migration, survival, and development of human hematopoietic cells. Increased expression levels of CXCL12 and CXCR4 induce proliferation of hematopoietic progenitors and recruitment of bone-resorbing osteoclasts, osteoblasts, neutrophils, and other myeloid cells, leading to leukocyte mobilization [7]. In addition, reduced CXCR4 expression levels might result in an amplified mobilization response and cell proliferation in the BM [8].

CD74 mRNA is expressed in HSPCs [9–11] however, the role of this receptor in HSPCs was never analyzed. CD74 is a type II integral membrane protein that is expressed many cell types. The CD74 chain was initially described to function mainly intracellularly as an MHC class II chaperone [12]. A small proportion of CD74 is modified by the addition of chondroitin sulfate (CD74-CS), and this form of CD74 is expressed on the cell surface. It was previously shown that macrophage migration inhibitory factor (MIF) binds to the CD74 extracellular domain, a process that results in the initiation of a signaling pathway [13].

MIF is also a noncognate ligand of the CXCRs, CXCR2, and CXCR4, and biochemical evidence suggests that these chemokine receptors could act as additional signal-transducing CD74 coreceptors upon MIF stimulation [14,15]. Importantly, structural and functional interactions between CD74 and the MIF chemokine receptor, CXCR4, have been proposed [14,16].

Our previous studies have shown that CD74 expressed on healthy and malignant B cells is directly involved in regulating murine mature B cell survival [17–20] through a pathway leading to the activation of transcription mediated by the NF-κB p65/RelA homodimer and its coactivator, TAFII105 [21]. NF-κB activation is mediated by the cytosolic region of CD74 (CD74-ICD), which is liberated from the membrane, and translocates to the nucleus [22]. Moreover, we demonstrated that CD74 stimulation by MIF enables augmented expression of antiapoptotic proteins in a CD44-dependent manner. In addition, we recently characterized the transcriptional activity of CD74-ICD. We showed that following CD74 activation, CD74-ICD interacts with the transcription factors RUNX and NF-κB and binds to proximal and distal regulatory sites enriched for genes involved in apoptosis, immune response, and cell migration. This leads to regulation of expression of these genes.

In the current study, the role of the MIF/CD74 axis in HSPCs was followed. We show that CD74 plays a crucial role in HSPC maintenance. Deficiency of CD74 and MIF leads to enhanced survival and accumulation of HSPCs in the BM. The enlarged pool of HSCs give rise to higher numbers of HSPCs and the various immune cell lineages. Cells lacking CD74 demonstrated an advantage in repopulating the host environment, as seen by the significantly higher levels of those cells when compared to the wild-type (WT) cells in mixed chimera. Thus, our study could lead to improved clinical insight into factors governing the efficacy of BM transplantation protocols, as well as diseases associated with hematopoietic failure.


Why are there heart-rate discrepancies in cycling vs. running

"There is an apparent difference between cycling and running heart rates," Monty writes. "Do you notice when you go out for an hour run at 85% of your max, it is just a good hard run, but when you try to ride at 85% you can last for about 10 minutes? Back in the old days we all just figured that that was the way it was, so what are you going to do?

"But Gary Hooker yes, the aero-bike-maker fellow did a lot of research on this kind of stuff, and as I pondered it, I found something very interesting. When Kenny Souza would ride and run in a race, his rate wouldn't vary but a couple beats in each discipline. Since he was the best in the world at the time at these two sports, I figured that he was doing something that the rest of us werent. It turns out that it isnt the heart that is the limiting factor in cycling. After more tests on world class cyclists-turned-triathletes, it turns out that they also have comparable heart rates in both disciplines.

"Gary figured out that it was just a function of strength-to-weight ratio. Most of us at the time were swimmers and runners, so we had never developed the necessary strength to push our hearts on the bike like we could running and swimming. So we were able to alter our training to include more weights and long, hard hill climbing, and eventually we were able to get those cycling heart rates closer to the running ones. The difference wasnt carved in stone like we thought."

Monty is, from a layman's view, right on in everything he says, with my picky exception regarding the causality. I don't think strength-to-weight ratio is the precise rationale. But it is entirely true that in general, cyclists can get their heart rates up during cycling much better than triathletes can when they ride the bike.

In fact I looked at a dozen or so studies this has been studied up the wazoo and in each case pure cyclists had fairly comparable heart rates riding the ergometer and running on a treadmill, whereas runners did fine on the treadmill but couldn't get their hearts up while on the bike. (Realize that in each case the athlete was able to achieve a higher heart rate while performing work in his given specialty.)

Road cycling is one of those odd sports, like speed skating and Nordic ski racing, in which aerobic capacity is obviously much needed, yet muscular power is almost as important. It may or may not be strength-to-weight, as Monty said, but cycling-specific power at any weight appears to be necessary.

But it isn't power at the top end that matters. One very smart physiologist with whom I correspond reminded me that Chris Boardman set a world hour record without being able to generate over 1,000 watts in a sprint and was unable to squat even a moderate amount of weight. But he could generate 440 watts for an entire hour. That sort of power is not necessarily gained through bigger muscles, but mostly through physiological changes at the cellular level.

Look at it this way. Swimming is an endurance activity just like running, yet swimmers are able to pull much more water than a runner-turned-triathlete. Yes, cycling and running are much more similar to each other than either are to swimming. The point is, cyclists develop their muscles in a sport-specific way that allows them to generate much more power cycling than a runner-turned-triathlete is able to.

There are several things you can do. Monty mentioned the most obvious, and that is to increase leg strength. Yes, lower-body weights is one way to go. I rather like his other option, which is hill riding. We are blessed in my area (north-inland San Diego County) because of our close proximity to many, and long, and steep inclines. You can pick your poison here: 20% grades or 4,000-foot grades. About the only thing San Diego doesn't have, where I ride, is a lack of grades.

Why is hill riding important? Because it forces an athlete to ride at a very high effort level for a sustained period of time. Let's face it, if you're toodling around town on the flats with your friends, your pulse isn't going to get up that high, and neither is your sustained power output. But what happens when you're climbing a 7% grade for several miles?

What I hope to impart is that it isn't set in stone that your heart rates must be 10 or 15 beats lower while on the bike vs. running. If you can't get your heart rate up while cycling it's simply because you're a better runner than a cyclist. The idea is not to attempt to raise your heart rate for the heck of it, but to raise the level of your cycling ability so that your well-trained cardiovascular system can get off the bench and into the game.

One way to achieve this is certainly to lift lower-body weights, as has been noted above. But I prefer more sport-specific ways, because that is why cyclists are able to generate cycling power that triathletes can't hope to match.

I'd prefer to perform power workouts while on the bike. Cyclists often motorpace for this, but there are logistical impediments to riding behind a powered scooter. One might resort to trainer workouts, such as the one I describe in another article on oxygen consumption drift. Another way is to ride a lot of hills, and specific hill workouts can be found on cycling-training sites throughout the Web.

There are two additional things I'll mention, both of which are controversial, and for which I have no scientific evidence to offer. They don't even rise to the level of hypotheses, and it might be best to style them "notions." Both center on the assumption that the better triathletes tend toward endurance physiology, i.e., they are less muscled, have predominantly slow-twitch muscle fibers, and they're geared toward endurance. Furthermore, they train themselves in a way that favors endurance physiology.

The art of competitive cycling rewards those who contain a curious admixture of abilities. Imagine a 10-mile footrace that occurs mostly at a steady jog, but with an occasional all-out sprint up a hill or along a quarter-mile stretch. Imagine that only those who keep up during these ballistic efforts are allowed to continue the race. It is apparent that power is crucial to top-level cycling.

In light of this, it is perhaps easier to imagine why a cyclist's leg power is so well-developed that when he rides a bicycle he can generate the same heart rates and oxygen consumption levels as when he runs.

What can be done for the poor run-trained slow-twitch triathlete? In addition to doing everything he can to increase his cycling power, there are some "work-arounds." One of these, in my view, is for a triathlete to ride with a steeper seat angle in conjunction with everything else we note in our articles on tri-bike fit.

In my view, this not only preserves a wider and more biomechanically efficient hip angle while riding in the aero position, it also allows him to spread out the work of the pedal stroke to other groups making up the hip musculature.

In other words, your peak power during the pedal stroke may be lower than a cyclist's, but your power application throughout the pedal stroke may be the same. Triathletes may never develop the huge vastus medialis muscles obvious on just about every pro cyclist, but his hamstrings may be able to take up much of the slack, and riding with a steeper seat angle make this easier.

Cadence also is critical to a triathlete's power, and this is not just an issue for triathletes. It's ironic that cyclists understand cadence and work at this to a degree triathletes don't.

In both examples above, triathletes will find themselves better able to work up to their endurance capacity, and to ride with higher heart rates without experiencing muscular exhaustion. Through a better use of cadence and "levers" they can trade in peak power which they have a hard time generating in any case for a more constant application of power, and it's application for more cycles over a given period of time.

In our discussion of the slow component of VO2 [see link above] one potential aggravator is the recruitment of fast-twitch muscle fibers when slow-twitch fibers are more appropriate for the work. I suspect that this is exacerbated by the application of higher peak power in place of sustained power, i.e., gear mashing instead of spinning.

This might lead to race phenomena like leg cramps even during the run segment that a triathlete might blame on electrolyte imbalance when he ought to look no further than his cycling training and technique.


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Materials and Methods

All mice were backcrossed to the C57Bl/6 background and were housed in a specific-pathogen-free animal facility, AALAC-accredited, at Baylor College of Medicine (Houston, TX).

Hematopoietic analyses

For peripheral blood analysis, transplant recipient mice (n = 8/genotype) were bled at 4, 8,12 and 16 weeks post transplantation. Red blood cells were lysed and samples were stained with CD45.1-APC, CD45.2-FITC, CD4-pacific blue, CD8-pacific blue, B220-pacific blue, B220-PE-cy7, Mac1-PE-cy7 and Gr-1-PE-cy-7 antibodies (BD Pharmingen, eBiosciences). FacsARIA, LSRII and FACS-Scan flow cytometers were used for analysis and cell sorting. Hematopoietic committed progenitors were analyzed based on expression of cell surface markers that can be identified using flow cytometry as described [37]. For complete blood counts, peripheral blood was collected from the retro-orbital plexus into tubes containing potassium EDTA (Sarstedt, Nümbrecht, Germany) from 8–10 week old WT and Atxn1L −/− mice (n = 10 mice/genotype) and analyzed with a Hemavet analyzer (Drew Scientific, TX, USA). LT-HSCs defined in text and legends. ST-HSCs: Sca-1 + , c-kit + , CD34 + , Flt3 − MPP: Sca-1 + , c-kit + , CD34 + , Flt3 + CLPs: Lineage − , IL7rα + , Sca-1 + , c-kit + CMP: Lineage − , IL7rα − , Sca-1 − , c-kit + , CD34 + , CD16/32 − GMP: Lineage − , IL7rα − , Sca-1 − , c-kit + , CD34 + , CD16/32 + MEP: : Lineage − , IL7rα − , Sca-1 − , c-kit + , CD34 − , CD16/32 − .

Bone marrow transplantation

Competitive bone marrow transplantation assays were performed by intravenous injection of admixed CD45.2 donor whole bone marrow cells with CD45.1 competitor bone marrow. Recipient C57Bl/6 mice had been lethally irradiated with a split dose of 10.5 Gy, 3 hours apart. Sex- and age-matched C57Bl/6 mice were used as competitors for every experiment. Eight recipient mice were used in each experiment (n = 8 mice/genotype), and each experiment was repeated at least twice. The competitor cell dose was kept constant at 250,000 cells in all transplants.

For the limiting dilution assays to determine repopulating units, we used 1.0×10 3 , 3.0×10 4 and 1.0×10 5 WT or Atxn1L −/− whole bone marrow cells pooled from (n = 3/genotype) CD45.2 sex- and age-matched mice (for the number of recipient animals in each dilution group, see Figure 2), mixed with 250,000 CD45.1 cells. Positive engraftment was scored based on multilineage repopulation of higher than 0.1%. The percentage of non-responders was calculated using the L-Calc software (StemCell Technologies).

HSC transplants were carried out as described above, but instead of whole bone marrow donor cells, purified HSCs were transplanted. Unless specified otherwise, HSCs were isolated using the side population (SP) method for Hoechst dye efflux [38], followed by KSL (c-Kit + , Sca1 + , lineage − ) and CD150 + staining. Recipient mice (n = 8/genotype) received 20 sorted HSCs and 250,000 WT competitor cells. Staining and isolation of HSCs were carried out as previously described [37].

For the secondary transplants, primary recipient mice were sacrificed 16 weeks post transplant and CD45.2 donor-derived HSCs were isolated as described above. Fifty HSCs were transplanted into secondary CD45.1 recipients, along with 250,000 CD45.1 competitor whole bone marrow.

Homing assay

Homing efficiency of donor cells into the recipient bone marrow was characterized in two ways. First, 30,000 KSL cells from pooled WT and Atxn1L −/− mice (CD45.2) were isolated and transplanted into lethally irradiated CD45.1 recipient mice (n = 5/genotype). Eighteen hours after the transplant, the recipient mice were sacrificed and their bone marrow was analyzed for the presence of CD45.2 positive cells using flow cytometry. A fraction of the whole bone marrow was also plated on methylcellulose medium in 32 mm dishes (n = 5 dishes/genotype). Controls included irradiated recipient mice that received no CD45.2 marrow. The resulting colonies are derived from the CD45.2 donor cells that homed into the recipient mice bone marrow. Thus, 12 days after plating, colonies were counted in each well to assess the homing efficiency of donor cells.

Methylcellulose cultures

HSCs were identified using the Hoechst dye efflux method along with positive staining for c-kit, Sca-1, CD150 and excluding lineage positive cells. Single HSCs were sorted in 96-well plates containing methylcellulose medium (StemCell Technologies). The number of colonies was counted at days 4, 7 and 14 and scored based on morphology on day 10.

For the in vitro colony proliferation assay, HSCs were sorted and plated into 6-well plates containing methylcellulose, 100 HSCs per well. Seven days later, single colonies were picked and resuspended in HBSS medium contain FBS (Gibco). The cell suspension was washed twice, stained with PI in sodium citrate and analyzed by flow cytometry.

Retroviral transduction

MSCV-Atxn1L-IRES-GFP and MSCV-IRES-GFP vectors were packaged using HEK293T cells by co-transfecting with pCL-Eco [39]. Mice were treated with 5-fluorouracil (150 mg/Kg body weight, American Pharmaceutical Partners) 6 days before harvesting the whole bone marrow. The bone marrow was enriched for Sca-1 expressing cells using magnetic selection (AutoMACS, Miltenyi), transduced with the retrovirus as previously described [7], and grown in culture. After 48 hours, cells were collected, stained for Sca-1, c-kit and lineage markers, and GFP+ KSL cells were sorted and plated into 6-well plates containing methylcellulose medium. Ten days later, the number of colonies in each well was counted.

Proliferation assays

Whole bone marrow from WT and Atxn1L −/− mice (n = 5/genotype) was isolated and stained for different hematopoietic progenitor populations. Cells were then fixed and stained for either BrdU or Ki-67 according to the BrdU staining protocol supplied by the manufacturer (BD-Pharmingen).

5-fluorouracil experiments

To determine how WT and Atxn1L −/− mice respond to stress, mice were treated with the chemotherapeutic drug, 5-FU. To determine the survival rate, mice (n = 5/genotype) received one injection of 5-FU (150 mg/Kg body weight) and analyzed for proliferation or apoptosis 3, 5 and 7 days later by flow cytometry.

In order to assess hematopoietic recovery after stress, WT and Atxn1L −/− mice (n = 10/genotype) were treated once with 5-FU and their peripheral blood counts were monitored every three days for 28 days using the Hemavet analyzer (Drew Scientific, TX, USA).

Annexin-V assays

Annexin-V staining was used to assess cell death and apoptosis. Briefly, cells were harvested and stained with the markers of interest according to the staining protocol described above. Cells were washed twice with cold PBS and incubated at room temperature in 1× binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2) containing Annexin V-APC (BD-Pharmingen). Cells were analyzed by flow cytometry within one hour of staining.

Microarray analysis

HSCs were purified as described above. Cells were purified from 8-week-old mice. Approximately 30,000 HSCs from WT and Atxn1L −/− mice were purified for RNA isolation. RNA was isolated using the RNAqueous kit (Ambion, Austin, TX, USA), and treated with DNase I. The RNA was linearly amplified using two rounds of T-7 based in vitro transcription using the MessageAmp kit (Ambion). The RNA was subsequently labeled with biotin-conjugated UTP and CTP (Enzo Biotech). The amplified RNA was hybridized to MOE430.2 chips according to standard protocol at the BCM Microarray core (Houston, TX). Data were analyzed by GCRMA with correction for false-discovery [40]. Data can be found in GEO, with accession number: GSE44285.

Bioinformatics analysis

In all cases of overlap analysis between gene sets, a Fisher's exact test was performed to determine p-values and statistical significance. We also generated Z-scores to measure the deviation between the observed overlap (number of genes in common between two sets) and what would be expected if one set were fixed and a random set was generated to overlap with it (eg. array overlap with P-sig, Q-sig and the HSC fingerprint). The expected overlap size was determined as the product between frequency of the fixed set and the size of the comparator set. The frequency was determined as the number of genes in the fixed set divided by the number of all genes that could be sampled the size of the comparator set was limited to the size of the comparator overlapped with the sample universe (i.e. when doing cross platform comparison, the comparator size was limited to the subset of the comparator represented among the universe of the fixed set. To generate the network in Figure 6D, we used homologene to map mouse gene symbols to human orthologs. We then identified all interactions in the interactome where one of the partners was expressed in HSC according to the HSC fingerprint dataset [7]. We used Cytoscape to generate the network image. We identified the gene products that were additionally identified as being HSC fingerprint genes by coloring them red. HSC expressed genes are colored pink.

For microarray data we used the R Bioconductor package GCRMA to process the low-level intensity data. We used the limma package to generate T-statistics and moderated p-values. We used the Bioconductor package mouse4302 v2.2 to determine the gene symbols for the probe sets on the array, and we used the same release to compare both the previously published HSC gene lists and our new array results.

Ethics statement

All animal work has been conducted according to national and international guidelines. The institutional animal care and use committee (IACUC) at Baylor College of Medicine approved the animal protocols for the work described herein. No human or primate samples were used for this work (data mining only).


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