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What do repopulation and expansion mean in stem cell biology?

What do repopulation and expansion mean in stem cell biology?


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I was studying a lecture on the effects of cytokines on hematopoiesis, and it uses these two terms often in the context of the effects of regulators on hematopoietic stem cells:

  • All repopulating bone marrow HSCs express a fibroblast growth factor (FGF) receptor that supports HSC expansion
  • TPO may has a role in promoting the survival of repopulating HSCs, however, it contributes to both generation and expansion of HSCs during definitive hematopoiesis.
  • Etc.

I looked online and honestly struggled to find any form of a definition on these two terms. They are used awfully alot though which makes it kinda weird too frankly.


Stem cells are defined as precursor cells that have the capacity to self-renew and to generate multiple mature cell types. Only after collecting and culturing tissues is it possible to classify cells according to this operational concept. This difficulty in identifying stem cells in situ, without any manipulation, limits the understanding of their true nature. This review aims at presenting, to health professionals interested in this area, an overview on the biology of embryonic and adult stem cells, and their therapeutic potential.

All the authors declared no competing interests.

TO CITE THIS ARTICLE: Chagastelles PC, Nardi NB. Biology of stem cells: an overview. Kidney inter., Suppl. 2011 1: 63–67.


A glossary for stem-cell biology

Stem-cell biology is in a phase of dynamic expansion and is forming connections with a broad range of basic and applied disciplines. The field is simultaneously exposed to public and political scrutiny. A common language in the stem-cell community is an important tool for coherent exposition to these diverse audiences, not least because certain terms in the stem-cell vocabulary are used differently in other fields.

Asymmetric division Generation of distinct fates in progeny from a single mitosis. Oriented division may position daughter cells in different microenvironments or intrinsic determinants may be segregated into only one daughter. Observed in some but not all stem cells and can occur in other types of progenitor cell.

Cancer cell of origin Precancerous cell that gives rise to a cancer stem cell. May be a mutated stem cell, or a committed progenitor that has acquired self-renewal capacity through mutation.

Cancer-initiating cell General term that encompasses both cancer cell of origin and cancer stem cell.

Cancer stem cell Self-renewing cell responsible for sustaining a cancer and for producing differentiated progeny that form the bulk of the cancer. Cancer stem cells identified in leukaemias and certain solid tumours are critical therapeutic targets.

Cell replacement therapy Reconstitution of tissue by functional incorporation of transplanted stem-cell progeny. Distinct from ‘bystander’ trophic, anti-inflammatory or immunomodulatory effects of introduced cells.

Clonal analysis Investigation of properties of single cells. Essential for formal demonstration of self-renewal and potency.

Commitment Engaging in a programme leading to differentiation. For a stem cell, this means exit from self-renewal.

Embryonic stem cell Pluripotent stem-cell lines derived from early embryos before formation of the tissue germ layers.

Founder/ancestor/precursor cell General terms for cell without self-renewal ability that contributes to tissue formation. In some cases they generate tissue stem cells.

Immortal strand The hypothesis of selective retention of parental DNA strands during asymmetric self-renewal. Potential mechanism to protect stem cells from the mutations associated with replication.

In vitro stem cell Self-renewal ex vivo in cells that do not overtly behave as stem cells in vivo. Occurs due to liberation from inductive commitment signals or by creation of a synthetic stem-cell state.

Label-retaining cell Candidate for adult tissue stem cell because of slow division rate and/or immortal strand retention. Interpret with caution.

Lineage priming Promiscuous expression in stem cells of genes associated with differentiation programmes.

Long-term reconstitution Lifelong renewal of tissue by transplanted cells. The definitive assay for haematopoietic, epidermal and spermatogonial stem cells. Transplantation assay may not be appropriate for all tissues.

Niche Cellular microenvironment providing support and stimuli necessary to sustain self-renewal.

Plasticity Unproven notion that tissue stem cells may broaden potency in response to physiological demands or insults.

Potency The range of commitment options available to a cell.

Totipotent Sufficient to form entire organism. Totipotency is seen in zygote and plant meristem cells not demonstrated for any vertebrate stem cell.

Pluripotent Able to form all the body's cell lineages, including germ cells, and some or even all extraembryonic cell types. Example: embryonic stem cells.

Multipotent Can form multiple lineages that constitute an entire tissue or tissues. Example: haematopoietic stem cells.

Oligopotent Able to form two or more lineages within a tissue. Example: a neural stem cell that can create a subset of neurons in the brain.

Unipotent Forms a single lineage. Example: spermatogonial stem cells.

Progenitor cell Generic term for any dividing cell with the capacity to differentiate. Includes putative stem cells in which self-renewal has not yet been demonstrated.

Regenerative medicine Reconstruction of diseased or injured tissue by activation of endogenous cells or by cell transplantation.

Reprogramming Increase in potency. Occurs naturally in regenerative organisms (dedifferentiation). Induced experimentally in mammalian cells by nuclear transfer, cell fusion, genetic manipulation or in vitro culture.

Self-renewal Cycles of division that repeatedly generate at least one daughter equivalent to the mother cell with latent capacity for differentiation. This is the defining property of stem cells.

Stem cell A cell that can continuously produce unaltered daughters and also has the ability to produce daughter cells that have different, more restricted properties.

Stem-cell homeostasis Persistence of tissue stem-cell pool throughout life. Requires balancing symmetric self-renewal with differentiative divisions at the population level, or sustained asymmetric self-renewal.

Stemness Unproven notion that different stem cells are regulated by common genes and mechanisms.

Tissue stem cell Derived from, or resident in, a fetal or adult tissue, with potency limited to cells of that tissue. These cells sustain turnover and repair throughout life in some tissues.

Transit-amplifying cell Proliferative stem-cell progeny fated for differentiation. Initially may not be committed and may retain self-renewal.


Materials and methods

Fetal liver (FL) cell collection and Western blot analysis

gp130 FXXQ/FXXQ and gp130 FXXQ/FXXQ STAT5ab ΔN/ΔN mice were generated by heterozygote crosses and PCR genotyping. E14.5 FL cells were collected as previously described from STAT5ab ΔN/ ΔN mice [14] and then stimulated with or without 50 ng/mL IL-6 for either 30 min. Western blot analysis was carried out as described [15].

FL cell transplantation and serial bone marrow transplants

FL cells were injected via the lateral tail-vein into lethally-irradiated primary recipients (1100 rads) either alone (non-competitive) or mixed 1:1 with CD45.1 FL cells (competitive) as described [14]. For non-competitive FL transplants, bone marrow was harvested from both hind limbs (tibias and femurs) of primary recipients and serially transplanted using at least 5 x 10 6 bone marrow cells (1 donor per 5 recipients). For limiting dilution competitive assays, FL cells were mixed with a radioprotective dose of 2휐 5 CD45.1 adult BM cells and injected via the lateral tail-vein into lethally-irradiated (1100 rads) adult Boy J mice.


Stem Cell Culture Basics

Stem cells require specialized, high-quality media and expert culture techniques for propagation in the laboratory. Suboptimal stem cell culture conditions can easily lead to unwanted stem cell differentiation or to cellular senescence. Stem cell differentiation is triggered by various factors in vivo, some of which can be replicated in in vitro stem cell cultures. Some stem cell lines are immortal and can be cultured indefinitely, so it is imperative to select the right stem cell type for your research application.

Recent advances in the stem cell field have been due to the advent of CRISPR genome editing technology and 3D cell culture techniques. Advanced protocols such as those that generate organoids from iPSCs have provided scientists with more predictive in vitro “disease-in-a-dish” models.


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Supplementary Figure 1 Subcutaneous and orthotopically grafted xenograft models are well differentiated.

(a) Hematoxylin and Eosin (H&E) staining of Co100 subcutaneous (s.c) xenograft. Scale bar, 500 μm. Representative of 5 independent tumours. (b) Immunofluorescent stainings of s.c. Co100 xenografts for mucin-2 (MUC2), cytokeratin 20 (CK20), intestine alkaline phosphatase (IAP), alpha smooth muscle actin (aSMA), lysozyme EC, alpha defensin-5 (aDef-5), all stainings (yellow), nuclear stain, Hoechst (blue). Scale bars, 50 μm (all panels). Representative of 5 independent tumours. (c and d) H&E, Alcian blue (c) and immunofluorescent F-actin (green) (d) staining for s.c. tumour models (Co100, CC09 and HCT-15), nuclear stain, Hoechst (blue). Scale bars, 50 μm (c) or 100 μm (d) Representative of 5 independent tumours. (e) H&E staining of Co100 and HCT-15 orthotopically grafted tumours (cecal wall). Scale bars, 500 μm. Representative of 3 independent tumours. (f) Orthotopically growing Co100 tumours do express differentiation markers as indicated (yellow). Nuclear stain, Hoechst (blue). Scale bars, 100 μm. Representative of 3 independent tumours

Supplementary Figure 2 In vitro and in vivo validation of LV-indLS2 system.

(a) Single cell cloned cultures of LV-indLS2 transduced cell lines were treated with increasing 4-OH-Tamoxifen (TAM) concentrations, mStrawberry expression was measured 7 days post induction by flow cytometry. (b) mStrawberry expression is randomly distributed amongst stem-like (TOP-GFP high ) or more differentiated (TOP-GFP low ) Co100.G7 cells directly after induction. (c) mStrawberry labeling is stable in time and does not influence clonal fitness. Single cell cloned LV-indLS2 transduced cultures of respectively Co100, HCT-15 and CC09 cells were treated with 4-OH-Tamoxifen or were cultured untreated as a control, mStrawberry expression was measured at day 7 and day 42 post-treatment by flow cytometry. (a-c) Data is represented as mean ± S.D., n = 3 independent experiments. n.s., not significant, two-sided Student’s t-test. (d) Confocal images of subcutaneous Co100 tumour xenografts, 7 days post induction with the indicated 4-OH-Tamoxifen concentrations. mStrawberry (red), nuclear staining, Hoechst (blue). Scale bars, 100 μm. Representatives of 3 tumours per group. (e) Percentage of mStrawberry + cells is dose-dependent, as determined by flow cytometry (n = 4 tumours per condition). (f, g) Confocal images of tumour xenograft sections were used for automated fluorescence image (FI) detection of mStrawberry + cells (f) and the percentage of single cell clones 7 days after induction was determined (g) (n = 10 tumour sections per condition). A 4-OH-Tamoxifen dose of 0.05 mg/mouse resulted in the induction of the optimal number of clones without evidence for clone collision the induction of too many clones results in overestimation of clone sizes. (e-g) Data is represented as mean ± S.D. (h) Quantification of the distances between cells from different clones 1 week after clonal induction with the indicated 4-OH-Tamoxifen concentrations. The red dashed lines indicate the maximum distance between cells within a clone

Supplementary Figure 3 Marker free lineage tracing and model inference for additional xenograft models.

(a, b) Images of labelled clones in Co100 tumours either orthotopically (a) or subcutaneously (b) grafted in NSG mice. Scale bars, 250 μm. (c, d) Representative images of labelled clones in HCT-15 (c) and CC09 (d) xenografts at indicated time points. mStrawberry (red), F-Actin (green), nuclear stain, Hoechst (blue). Scale bars, 250 μm. (e-h) Sectional clone size distribution over time in orthotopic Co100 (e), subcutaneous Co100 (NSG mice) (f), HCT-15 (g) and CC09 (h) xenografts. Depicted are the experimentally measured fractions of growing clones within the indicated bins (black dots) and the model predicted clone size distributions using the optimal fit parameters (dashed line). Data is represented as mean ± S.E.M., grey shade represents 95% confidence interval of prediction. Source data for Supplementary Figure 3 can be found in Supplementary Table 1

Supplementary Figure 4 Proliferation is associated with stromal cells but not with vasculature or hypoxia.

(a) Images of human primary colorectal cancer tissue. Ki67 + cells, (yellow), myofibroblasts (aSMA, green). Nuclear stain, Hoechst (blue). Scale bars, 0.5 mm (left) and 100 μm (right), representative of 10 independent tumours. (b) Percentage of Ki67 + cells is significantly higher in the edge region (E) of human primary colon cancer tissue compared to centre (C). (c) Average distance of either Ki67 negative cells or proliferating (Ki67 + ) tumour cells to the nearest aSMA + fibroblast in human primary colon cancer tissue. (b, c) Data is represented as mean ± S.D., n = 10 tumours. (Two-tailed Wilcoxon matched pairs signed rank test). (d-e) Cleaved Caspase-3 (green) (d) or CD31 (yellow) (e) expression in edge (upper) and centre areas (lower) in a Co100 tumour section. Nuclear staining, Hoechst (blue). Scale bars, 100 μm (d) or 200 μm (e), representative of 5 tumours. (f) Immunohistochemical staining for HIF-1a in the edge (upper) and centre (lower) of a Co100 tumour. Scale bars, 100 μm, representative of 3 tumours. (g) Gene set enrichment analysis on RNA expression profiles of cells located at the edge and centre of the xenografts indicates an enrichment for DNA replication and cell cycle gene sets (Molecular Signatures Database entries M1017 and M5468) in the edge region, but not for hypoxia associated genes 43 , n = 2 tumours per cell line. (h) Immunofluorescence staining for aSMA (green) and Ki67 (yellow), in HCT-15 (upper panel) and CC09 (lower panel) xenografts. Nuclear stain, Hoechst (blue). Scale bars, 100 μm. Right images are magnifications of the box in the left images. (i) Average distance of either all or proliferating (Ki67 + ) tumour cells to the nearest aSMA + fibroblast in HCT-15 and CC09 (n = 20.000 cells from 6 tumours per cell line) xenografts. (j) Average distance of either all or apoptotic (CC3 + ) tumour cells to the nearest aSMA + fibroblast in Co100 tumours treated with Oxaliplatin-5FU (n = 20.000 cells from 4 tumours). (i, j) Data is represented as mean ± S.E.M., distances were compared using paired two-tailed Student’s t-test

Supplementary Figure 5 Growth of individual tumour xenografts is best fitted using a surface growth model.

(a) Tumour growth of individual subcutaneous Co100, HCT-15 and CC09 tumour xenografts was measured in time (black dots). Solid and dashed lines represent respectively model fits of exponential volume and surface growth. Both models are fitted with the initial volume constrained between 0 and the total volume of injected cells (0.1 mm 3 ). (b) Goodness of fit measured by R 2 (R-squared), comparing the surface and exponential volume growth models indicate a better fit of the surface model for almost all tumours. R 2 values smaller than zero are rounded up to zero. (c) The Akaike information criterion (AIC) similarly indicated surface growth as the best model to describe the data (the best model has the lowest AIC value). (a-c) N = 29 (Co100), 25 (HCT-15) and 23 (CC09) tumours

Supplementary Figure 6 Osteopontin is a fibroblast secreted factor that enhances clonogenicity.

(a) Co100 cells were adherently seeded as single cells, with or without human or mouse primary intestinal fibroblasts conditioned medium. 3 days after seeding, cells were stained for F-Actin (green) and nuclear stain Hoechst (blue). Scale bars, 100 μm, representative of 10 images per condition. (b) Quantification of clone sizes of data shown in panel (a), n = 10 images per condition. (c) Proliferation of Co100 cells growing in a mixture of reduced growth medium and fibroblast conditioned medium, 3 days after seeding, n = 3 replicates, data is represented as mean ± S.D. (d) Analysis of murine gene expression, specifically encoding secreted proteins, obtained from RNAseq data of 3 different xenograft models, revealed Spp1, encoding osteopontin (OPN) as the most abundantly expressed. (e) In vitro treatment of Co100 cells with recombinant human OPN (500 ng/ml) increased proliferation, n = 3 independent experiments. (f, g) Analysis of OPN expression of Co100.OPN cells on mRNA (n = 1 experiment with 3 technical replicates) (f) and secreted protein levels (n = 2 independent experiments) (g) by respectively qPCR and ELISA. (wt, wildtype Co100, neg, OPN - Co100 population, OPN, OPN + Co100 population) (e-g) Data is represented as mean ± S.D. (h) Relative clone frequency (colour) per clone size (columns) in time (rows) for Co100.OPN tumours. Number of clones and tumours (between parentheses) are depicted. (i) Goodness of fit (inverse and normalized least-squares distance) as a function of A and h on expanding clones (clone size > 1 cell) in Co100.OPN xenografts. Source data are shown in Supplementary Table 1. (j) Ki67 (yellow) expression in Co100.OPN xenografts, aSMA (green), nuclear stain, Hoechst (blue). Scale bars, 500 μm, representative of 8 tumours per group. Quantification of Ki67 (k) and aSMA (l) expression in Co100 (white bars) (aSMA as shown in Fig. 5h) and Co100.OPN (red bars) xenografts. (n = 8 (Co100) and 12 (Co100.OPN) for Ki67 and n = 10 (Co100) and 12 (Co100.OPN) for aSMA . Two-tailed Student’s t-test, to compare edge to centre a paired two-tailed Student’s t-test was used. Data is represented as mean ± S.D

Supplementary Figure 7 Example figure FACS gating strategy.

Cells were selected in FSC/SSC dot plot to remove debris, single cells were gated using the FSC-H/FSC-W dot plot. GFP+, mStrawberry+ or PE+ cells were gated and compared with a control sample with no detectable fluorochrome expression


Discussion

Prompted by the surprising observation of a power law dependence of T cell fold expansion on initial cell numbers (3), we developed a simple mathematical model in which T cell proliferation is stimulated by a dynamically changing number of cognate pMHC molecules and showed that it naturally yields the observed power law. We then explored more generally how T cell numbers, TCR affinity for antigen, and the dynamics of pMHC presentation combine to regulate T cell expansion in different regimes. Testing these results against other experimental datasets, we found that our model correctly predicts a power law dependence of T cell expansion on affinity for low-affinity antigens (6), a quantitative relationship that had not previously been appreciated. With regard to vaccination, our model furthermore predicts the observed enhanced efficacy of stretching a fixed total antigen dose over several days (7) and provides testable predictions for how to optimize antigen dosing.

The core of our model is that T cell expansion is regulated by dynamically changing levels of presented antigens. The dynamics of pMHCs is assumed to be characterized by a fast processing of antigens by antigen-presenting cells followed by a slower decay. The first assumption of rapid processing seems well justified for the subcutaneous injection of antigens used in ref. 3 but might be more questionable in ref. 6, where live replicating bacteria are used, as this might lead to continued processing of new antigens by dendritic cells. However, as the bacterial load declines rapidly after reaching a peak at 3 d postinfection (17, 18), newly generated pMHCs likely play a small role compared with the turnover of the already presented pMHCs at the late stages of infection analyzed in Fig. 2. The second assumption of the decay of presented pMHCs is experimentally well supported (8, 9) and has a known mechanistic basis (12, 13). Furthermore, our inferred decay constants are within the range of decay constants reported for different antigens bound to dendritic cells (8) and are also roughly compatible with direct measurements of the decrease of the stimulatory capacity of antigen-presenting cells in transgenic mice after switching off inducible antigen production (9). One limitation of our model, particularly for replicating antigens, is that we have neglected any influence of the epitope-specific pMHC density on the surface of an antigen-presenting cell, which may also play a role in determining T cell stimulation and expansion (19).

The kinetics of antigen administration has been shown to influence the magnitude of T cell (7) and B cell (20) immune responses. This finding has implications for the rational design of vaccination strategies (21), but open questions remain regarding how to optimize dosing for high magnitude of response and/or high affinity of the responding cells (20). Our modeling suggests that exponentially increasing doses are close to optimal for maximizing the magnitude of T cell response. We further find that selection for higher affinity is strongest when T cells compete for antigen stimulation. Selection for affinity is thus predicted to be more stringent for lower or more slowly increasing antigen levels and for larger or faster growing prior T cell populations. This could have important implications for patterns of immunodominance in primary vs. secondary infections: In secondary infections, competition is expected to be stronger, as preexisting memory cells specific to the pathogen are usually present in higher numbers and can also proliferate faster.

Looking ahead, our model could be further extended to make it more realistic. Including single-cell stochasticity could help clarify how reproducible population-level expansion arises despite stochasticity at the single-cell level (22) and would also allow connections to recent experimental studies, which have revealed substantial heterogeneity of the immune responses of single cells (23). Furthermore, the model could be extended to account for the diverse compartments (different lymph nodes, the spleen, different tissues) (2) and T cell subtypes (4) involved in an immune response. Spatial or cellular heterogeneity can create separate niches in which T cells compete for proliferation and survival, which may provide another layer of regulation of T cell expansion.

Among the open questions raised in our study, we highlight two. First, other than regulating T cell expansion during an acute infection, how else might antigen presentation levels influence T cell populations, and do they play a role in thymic selection or the dynamics of naive T cells competing for self-antigens? Specifically, recent work in ecology (24) suggests that T cell grazing, which consumes antigens, could allow for the coexistence of a diverse naive repertoire despite competition for self-antigens. Second, regulation of T cell population dynamics can be achieved by tuning of system parameters. For example, smart control of the lifetime of presented pMHC complexes could induce the “right” amount of T cell amplification. Might system parameters have evolved to be close to optimal, and/or could some parameters also be regulated during an immune response to provide a robust response to infections while avoiding autoimmunity?


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Part I: Introduction to Stem Cells

Chapter 1. Why Stem Cell Research? Advances in the Field

1.1 The Origins of Stem Cell Technology

1.2 Organizations that Advocate and Support the Growth of the Stem Cell Sector

1.3 Applications of Stem Cells in Medicine

1.4 Challenges to the Use of Stem Cells

Chapter 2. ‘Stemness’: Definitions, Criteria, and Standards

2.6 Where do Stem Cells Come from?

2.7 Stem Cells of the Early Embryo

2.8 Ontogeny of Adult Stem Cells

2.9 How are Stem Cells Identified, Isolated, and Characterized?

2.12 Stemness: Progress Toward a Molecular Definition of Stem Cells

Chapter 3. Pluripotent Stem Cells from Vertebrate Embryos: Present Perspective and Future Challenges

3.2 Biology of ES and ESL Cells

Chapter 4. Embryonic Stem Cells in Perspective

4.1 Embryonic Stem Cells in Perspective

Chapter 5. The Development of Epithelial Stem Cell Concepts

5.2 A Definition of Stem Cells

5.3 Hierarchically Organized Stem Cell Populations

5.5 The Intestinal Stem Cell System

5.6 Stem Cell Organization on the Tongue

Part II: Basic Biology and Mechanisms

Chapter 6. Stem Cell Niches

6.1 Stem Cell Niche Hypothesis

6.2 Stem Cell Niches in the Drosophila Germ-Line

6.3 The Germ-Line Stem Cell Niche in the Drosophila Ovary

6.4 Germ-Line Stem Cell Niche in the Drosophila Testis

6.5 Coordinate Control of Germ-Line Stem Cell and Somatic Stem Cell Maintenance and Proliferation

6.6 Structural Components of the Niche

6.7 Stem Cell Niches Within Mammalian Tissues

Chapter 7. Mechanisms of Stem Cell Self-Renewal

7.1 Self-Renewal of Pluripotent Stem Cells

7.2 Prevention of Differentiation

7.3 Maintenance of Stem Cell Proliferation

7.4 Maintenance of Telomere Length

7.5 X Chromosome Inactivation

Chapter 8. Cell Cycle Regulators in Stem Cells

8.2 Cell Cycle Kinetics of Stem Cells In Vivo

8.3 Stem Cell Expansion Ex Vivo

8.4 Mammalian Cell Cycle Regulation and Cyclin-Dependent Kinase Inhibitors

8.5 Roles of Cyclin-Dependent Kinase Inhibitors in Stem Cell Regulation

8.6 Roles of p21 in Stem Cell Regulation

8.7 Roles of p27 in Stem Cell Regulation

8.8 Other Cyclin-Dependent Kinase Inhibitors and the Retinoblastoma Pathway in Stem Cell Regulation

8.9 Relation Between Cyclin-Dependent Kinase Inhibitors and Transforming Growth Factor β-1

8.11 Summary and Future Directions

Chapter 9. How Cells Change Their Phenotype

9.1 Metaplasia and Transdifferentiation

9.2 Examples of Transdifferentiation

9.5 Bone Marrow to Other Cell Types

9.6 Dedifferentiation as a Prerequisite for Transdifferentiation

9.7 How to Change a Cell’s Phenotype Experimentally

Part III: Tissue and Organ Development

Chapter 10. Differentiation in Early Development

10.1 Preimplantation Development

10.2 Cell Polarization Occurs During Compaction

10.3 Axis Specification During Preimplantation in the Mouse

10.4 Developmental Potency of the Early Mouse Embryo

10.5 Genes Important During Preimplantation Mouse Development

10.6 From Implantation to Gastrulation

10.7 The Mouse Trophectoderm and Primitive Endoderm Cells

10.8 Development of the Mouse Inner Cell Mass to the Epiblast

10.10 Implantation: Maternal Versus Embryonic Factors

10.11 The Role of Extra-Embryonic Tissues in Patterning the Mouse Embryo

Chapter 11. Stem Cells Derived from Amniotic Fluid

11.1 Amniotic Fluid – Function, Origin, and Composition

11.2 Amniotic Fluid Mesenchymal Stem Cells

11.3 Amniotic Fluid Stem Cells

Chapter 12. Stem and Progenitor Cells Isolated from Cord Blood

12.1 Addressing Delayed Time to Engraftment and Graft Failure With CB

12.2 Cryopreservation of CB Cells

12.3 Induced Pluripotent Stem Cells Generated from CB

Chapter 13. The Nervous System

13.4 Neural Differentiation of Mouse ES Cells

13.5 Neural Differentiation of Human and Nonhuman Primate ES Cells

13.6 Developmental Perspectives

13.7 Therapeutic Perspectives

Chapter 14. Sensory Epithelium of the Eye and Ear

14.2 Introduction to Progenitor and Stem Cells in the Retina

14.3 The Optic Vesicle Generates Diverse Cell Types that can Undergo Transdifferentiation

14.4 In Vivo Neurogenesis in the Posthatch Chicken

14.5 Growth of Retinal Neurospheres from the Ciliary Margin of Mammals

14.6 Prospects for Stem Cell Therapy in the Retina

14.7 Development and Regeneration of Tissues Derived from the Inner Ear

14.8 In Vivo Neurogenesis in Postembryonic Animals

14.9 In Vitro Expansion of Otic Progenitors

14.10 Prospects for Therapy

Chapter 15. Epithelial Skin Stem Cells

15.1 A Brief Introduction to Mouse Skin Organization

15.2 The Bulge as a Residence of Epithelial Skin Stem Cells

15.3 Models of Epithelial Stem Cell Activation

15.4 Molecular Fingerprint of the Bulge – Putative Stem Cell Markers

15.5 Cell Signaling in Multipotent Epithelial Skin Stem Cells

15.6 Commentary and Future Directions

Chapter 16. Hematopoietic Stem Cells

16.1 Embryonic Stem Cells and Embryonic Hematopoiesis

16.2 Blood Formation in Embryoid Bodies

16.3 Transformation of an EB-Derived HSC by BCR/ABL

16.4 Promoting Hematopoietic Engraftment with STAT5 and HOXB4

16.5 Promoting Blood Formation In Vitro with Embryonic Morphogens

Chapter 17. Peripheral Blood Stem Cells

17.2 Types and Source of Stem Cells in the Peripheral Blood

17.3 Endothelial Progenitor Cells

17.4 Mesenchymal Stem Cells

17.5 Therapeutic Applications of Peripheral Blood Stem Cells

17.6 Conclusions and Future Directions

Chapter 18. Multipotent Adult Progenitor Cells

18.1 Pluripotent Stem Cells – Embryonic Stem Cells

18.2 Postnatal Tissue-Specific Stem Cells – Are Some More than Multipotent?

18.3 Can Pluripotency Be Acquired?

18.4 Isolation of Rodent MAPCs

18.5 Isolation of Human MAPCs

Chapter 19. Mesenchymal Stem Cells

19.1 The Definition of MSCs

19.2 The Stem Cell Nature of MSCs

19.3 Which Tissues Contain MSCS?

19.4 MSC Isolation Techniques

19.5 Immunomodulatory Effects of MSCS

19.6 Skeletal Tissue Regeneration by MSCS

19.7 Non-Skeletal Tissue Regeneration by MSCS

Chapter 20. Skeletal Muscle Stem Cells

20.2 The Original Muscle Stem Cell: The Satellite Cell

20.3 Functional and Biochemical Heterogeneity Among Muscle Stem Cells

20.4 Unorthodox Origins of Skeletal Muscle

20.5 The Muscle Stem Cell Niche

Chapter 21. Stem Cells and the Regenerating Heart

21.2 Recruiting Circulating Stem Cell Reserves

21.3 The Elusive Cardiac Stem Cell

21.4 Evolving Concepts of Regeneration

Chapter 22. Cell Lineages and Stem Cells in the Embryonic Kidney

22.1 The Anatomy of Kidney Development

22.2 Genes that Control Early Kidney Development

22.3 The Establishment of Additional Cell Lineages

22.4 What Constitutes a Renal Stem Cell?

Chapter 23. Adult Liver Stem Cells

23.1 Organization and Functions of Adult Mammalian Liver

Chapter 24. Pancreatic Stem Cells

24.2 Definition of Stem Cells and of Progenitor Cells

24.3 Progenitor Cells During Embryonic Development of the Pancreas

24.4 Progenitor Cells in the Adult Pancreas

24.5 Forcing Other Tissues to Adopt a Pancreatic Phenotype

Chapter 25. Stem Cells in the Gastrointestinal Tract

25.2 Gastrointestinal Mucosa Contains Multiple Lineages

25.3 Epithelial Cell Lineages Originate from a Common Precursor Cell

25.4 Single Intestinal Stem Cells Regenerate Whole Crypts Containing all Epithelial Lineages

25.5 Mouse Aggregation Chimeras Show that Intestinal Crypts are Clonal Populations

25.6 Somatic Mutations in Stem Cells Reveal Stem Cell Hierarchy and Clonal Succession

25.7 Human Intestinal Crypts Contain Multiple Epithelial Cell Lineages Derived from a Single Stem Cell

25.8 Bone Marrow Stem Cells Contribute to Gut Repopulation After Damage

25.9 Gastrointestinal Stem Cells Occupy a Niche Maintained by ISEMFs in the Lamina Propria

25.10 Multiple Molecules Regulate Gastrointestinal Development, Proliferation, and Differentiation

25.11 Wnt/β-Catenin Signaling Pathway Controls Intestinal Stem Cell Function

25.12 Transcription Factors Define Regional Gut Specification and Intestinal Stem Cell Fate

25.13 Gastrointestinal Neoplasms Originate in Stem Cell Populations

Chapter 26. Induced Pluripotent Stem Cells

26.1 Generation of iPS Cells

26.2 Molecular Mechanisms in iPS Cell Induction

26.3 Recapitulation of Disease Ontology and Drug Screening

26.5 Safety Concerns for Medical Application

Chapter 27. Embryonic Stem Cells: Derivation and Properties

27.1 Derivation of Embryonic Stem Cells

27.2 Culture of Embryonic Stem Cells

27.3 Developmental Potential of Embryonic Stem Cells

Chapter 28. Isolation and Maintenance of Murine Embryonic Stem Cells

28.2 Maintenance of Embryonic Stem Cells

28.5 Colony-Forming Assay for Testing Culture Conditions

28.6 Embryonic Stem Cell Passage Culture

28.7 Isolation of New Embryonic STEM Cell Lines

28.8 Method for Deriving Embryonic Stem Cells

Chapter 29. Approaches for Derivation and Maintenance of Human Embryonic Stem Cells: Detailed Procedures and Alternatives

29.3 Preparing and Screening Reagents

29.4 Mechanical Passaging of hES Cell Colonies

29.5 Derivation of hES Cells

29.6 Maintenance of Established hES Cell Cultures

29.9 hES Cell Quality Control

Chapter 30. Derivation and Differentiation of Human Embryonic Germ Cells

30.2 Human Embryonic Germ Cell Derivation

30.3 Embryoid Body-Derived Cells

Chapter 31. Genomic Reprogramming

31.2 Genomic Reprogramming in Germ Cells

31.3 Reprogramming Somatic Nuclei

Chapter 32. Neural Stem Cells – Therapeutic Applications in Neurodegenerative Diseases

32.2 Definition of Neural Stem Cells

32.3 Therapeutic Potential of Neural Stem Cells

32.4 Gene Therapy Using Neural Stem Cells

32.5 Cell Replacement Using Neural Stem Cells

32.6 ‘Global’ Cell Replacement Using Neural Stem Cells

32.7 Neural Stem Cells Display an Inherent Mechanism for Rescuing Dysfunctional Neurons

32.8 Neural Stem Cells as the Glue That Holds Multiple Therapies Together

Chapter 33. Adult Progenitor Cells as a Potential Treatment for Diabetes

33.1 Importance of β-Cell Replacement Therapy for Diabetes and the Shortage of Insulin-Producing Cells

33.2 Potential of Adult Stem-Progenitor Cells as a Source of Insulin-Producing Cells

33.3 Defining β-Cells, Stem Cells, and Progenitor Cells

33.4 New β-Cells are Formed Throughout Adult Life

33.5 What is the Cellular Origin of Adult Islet Neogenesis?

33.6 Transdifferentiation of Nonislet Cells to Islet Cells

33.7 Pancreatic Acinar Cell Transdifferentiation

33.8 Bone Marrow Cells as a Source of Insulin-Producing Cells

33.9 Liver as a Source of Insulin-Producing Cells

33.10 Engineering Other Non-β-Cells to Produce Insulin

33.11 Attempts to Deliver Insulin Through Constitutive Rather Than Regulated Secretion

Chapter 34. Burns and Skin Ulcers

34.2 Burns and Skin Ulcers – The Problem

34.4 Stem Cells in Burns and Skin Ulcers – Current Use

34.5 Recent and Future Developments

Chapter 35. Stem Cells and Heart Disease

35.1 Heart: A Self-renewing Organ

35.2 Distribution of CSCS in the Heart

35.3 Repair of Myocardial Damage by Nonresident Primitive Cells

35.4 Repair of Myocardial Damage by Resident Primitive Cells

35.5 Myocardial Regeneration in Humans

Chapter 36. Stem Cells for the Treatment of Muscular Dystrophy

36.2 Myoblast Transplantation – Past Failure and New Hope

36.3 Unconventional Myogenic Progenitors

36.4 Pluripotent Stem Cells for Future Cell-Based Therapies

Chapter 37. Cell Therapy for Liver Disease: From Hepatocytes to Stem Cells

37.3 Integration of Hepatocytes Following Transplantation

37.4 Clinical Hepatocyte Transplantation

37.6 Hepatocyte Transplantation in Acute Liver Failure

37.7 Hepatocyte Transplantation for Metabolic Liver Disease

37.8 Hepatocyte Transplantation – Novel Uses, Challenges, and Future Directions

Chapter 38. Orthopedic Applications of Stem Cells

38.5 Ligaments and Tendons

Chapter 39. Embryonic Stem Cells in Tissue Engineering

39.2 Tissue Engineering Principles and Perspectives

39.3 Limitations and Hurdles of Using ES Cells in Tissue Engineering

Part VI: Regulation and Ethics

Chapter 40. Ethical Considerations

40.2 Is it Morally Permissible to Destroy a Human Embryo?

40.3 Should we Postpone hES Cell Research?

40.4 Can We Benefit from Others’ Destruction of Embryos?

40.5 Can We Create an Embryo to Destroy it?

40.6 Should We Clone Human Embryos?

40.7 What Ethical Guidelines Should Govern hES Cell and Therapeutic Cloning Research?

Chapter 41. Overview of the FDA Regulatory Process

41.1 Introduction and Chapter Overview

41.2 Brief Legislative History of FDA

41.3 Laws, Regulations, and Guidance

41.4 FDA Organization and Jurisdictional Issues

41.5 Approval Mechanisms and Clinical Studies

41.6 Meetings with Industry, Professional Groups, and Sponsors

41.7 Regulations and Guidance of Special Interest for Regenerative Medicine

41.8 FDA’s Standards Development Program

41.9 Advisory Committee Meetings

41.10 FDA Research and Critical Path Science

41.11 Other Communication Efforts

Chapter 42. It’s Not about Curiosity, It’s about Cures: Stem Cell Research – People Help Drive Progress

42.3 Personal Promises Fuel Progress

42.6 People Drive Progress

42.7 Better Health for All


Growth Factors in Stem Cell Biology

Stem cell biology researchers use suitable growth factors to trigger proliferation, differentiation and/or migration of stem cells. Embryonic pluripotent stem cells can differentiate into three germ layers (endoderm, mesoderm, and ectoderm) and unlimited capacity for self-renewal 3 . The ethical issues around the use of embryonic stem cells led to the introduction of induced pluripotent stem cells or iPSCs. In the presence of growth factors, iPSCs differentiate into majority of the progenitor cells required for development (Table 1). Therefore, the role of growth factors in differentiation of iPSCs provides an avenue for creating an unlimited supply of embryonic-like stem cells (Figure 1).

Figure 1. iPSCs differentiate into majority of the progenitor cells required for development in the presence of Growth Factors

The fate of pluripotent stem cell is Stem cell research is controlled by physical and biochemical cues that direct them to become the specialized cells that make up the tissues in the body. Stem cell research is enhancing our understanding of how growth factors (biochemical cues) affect stem cell expansion and differentiation. This will enable subsequent use of stem cells in cell-based therapies, drug development, and disease modeling.


4 DISCUSSION

β-Catenin/canonical Wnt signaling has been shown to be important for the emergence of the first functional HSCs 7, 18 but its role in regulating HSCs in the fetal liver has been largely unexplored. We compared here the potential for canonical and noncanonical Wnt signaling in fetal and adult CD150 + LSKs using gene/protein expression as well as functional measures. Our data indicate that canonical Wnt signaling is predominantly active in fetal HSPCs, whereas both adult and fetal HSPCs have the potential to activate noncanonical pathways. We further suggest that β-catenin regulates the metabolism and short-term competitiveness of fetal HSPCs after transplant, and that the switch from fetal to adult HSCs corresponds to a downregulation of β-catenin/canonical Wnt signaling.

The specific role of canonical Wnt signaling in adult HSCs still remains under debate, and is likely to depend on dose and situation. Highly proliferative cells, such as leukemic cells, depend on β-catenin, 9, 45 while adult HSCs are maintained in the niche by noncanonical Wnt signaling and activate β-catenin only upon stimulation. 10, 14, 15 Our results indicate that higher levels of β-catenin and/or canonical Wnt signaling are required for maintaining short-term HSC function in the FL than in the adult BM. This also supports the idea that a specific gradient of β-catenin is required for HSC function at each stage of development, and that highly proliferative cells may be more dependent on β-catenin than quiescent cells. However, β-catenin was not responsible for promoting proliferation, but appeared rather to have an early protective effect as shown by increased ROS accumulation in Ctnnb1 Δ/Δ HSPCs. This difference in ROS levels was not present in pretransplant FL HSPCs, which were overall higher than what was observed in Ctnnb1 fl/fl HSPCs in the posttransplant BM but fairly comparable to posttransplant Ctnnb1 Δ/Δ HSPCs. These findings suggest that the increased ROS levels detected in Ctnnb1 Δ/Δ HSPCs could be interpreted as enhanced metabolic activity due to lack of quiescence or an inadequate association with hypoxic niches. Although β-catenin was dispensable for HSPC homing to the BM, it may promote early engraftment by other mechanisms, such as association with BM niches, which ultimately result in enhanced FL HSPC expansion early after transplant. It is also interesting that we did not detect any differences in long-term transplants and Ctnnb1 Δ/Δ HSPCs were “rescued” after the initial burst of HSPC expansion. This could be due to lower Cebpa expression that has been associated with improved long-term repopulation ability, 36 and further suggests that the decrease in Ctnnb1 Δ/Δ donor FL HSPCs detected in the bone marrow 6 weeks posttransplant may be more likely due to a difference in initial homing and engraftment in the absence of β-catenin, rather than actual defects in proliferation and expansion.

We observed a significant difference in the proportion of FL CD150 + LSKs staining positive for active (nonphosphorylated) β-catenin (Figure 2B and C) as compared to those expressing the H2B-GFP reporter (Figure 2D). There are several possible explanations for this discrepancy. For one, the fact that FL HSCs are actively proliferating could decrease the sensitivity of the reporter assay, and the mice used for the assay were hemizygous. H2B-GFP + HSPCs would thus represent only those cells that had recently received the signal and had not yet divided. Alternatively, but not exclusively, FL β-catenin could also form other, TCF/LEF-independent, transcriptional complexes, such as the YAP complex, 46 HIF-1α, 47 or Foxo, 48 as well as be found associated with membrane-bound adhesion complexes. 49, 50 Furthermore, we cannot fully exclude the possibility that Wnt/β-catenin signaling is restricted to the early phases of FL HSPC expansion and that slight differences in fetal developmental stage could make a major difference (e13 vs. e13.5–e14, a difference that is fully conceivable between two different experiments, especially when using two different strains of mice).

It has been reported that canonical Wnt signaling is downregulated during HSC aging. 29 Our findings suggest that this switch from canonical to noncanonical Wnt signaling begins already during the transition from fetal to adult stage. Fetal HSCs acquire the characteristics of adult HSCs, including quiescence, within 6 weeks after transplant. 22 The fact that we observed no long-term defect in our transplant recipients also argues for a selective, transient role of β-catenin-dependent canonical Wnt signaling in HSC ontogeny as previously suggested. 7 It also suggests that there are likely to be profound differences in Wnt ligand availability between FL and adult BM microenvironments. Indeed, comparisons between mesenchymal stromal cells isolated from FL and BM appear to confirm this hypothesis. 51

Adult HSCs give a balanced lineage output at steady state and after transplant. Interestingly the impact of canonical Wnt signaling appears to be lineage-specific, with T-cell differentiation being associated with more elevated levels of canonical Wnt/β-catenin signaling than myeloid cell differentiation or HSC self-renewal. 11, 52 In parallel, non-canonical Wnt5a-dependent signaling has been associated with increased thymocyte apoptosis. 52, 53 We observed a delay in myeloid and B lymphoid reconstitution by Ctnnb1 Δ/Δ FL cells together with a trend toward delayed T lymphoid development, suggesting that the absence of β-catenin had little impact on lineage bias in FL HSPCs. However, our observation regarding elevated canonical Wnt signaling activity in FL HSCs dovetails well with the decreased frequency of myeloid-biased fetal HSCs when compared to adult or aged BM. 54, 55 It is also of note that an age-associated decrease in canonical Wnt signaling has been reported in human HSCs and early T-progenitor cell subsets, where it correlated with deficient T lymphocyte output. 56

Altogether, moderate levels of canonical Wnt/β-catenin signaling appear to contribute to a balanced lympho-myeloid output while Wnt5a/Cdc42-dependent signals favor myeloid differentiation at the expense of T lymphocytes. However, only little is known at present about the potential role of other noncanonical signaling pathways. 57-59 Most noncanonical Frizzled receptors and coreceptors were expressed at the mRNA level by both FL and adult BM HSCs in our assay and their respective roles will warrant further study to better delineate the different branches of Wnt signaling in hematopoietic cells.

In summary, we have demonstrated here that the most prominent Wnt signaling pathways differ between fetal and adult CD150 + LSKs at steady state and that the early expansion of fetal HSPCs after transplant is dependent on β-catenin. These findings will further our understanding of the mechanisms that underlie fetal HSPCs’ enhanced reconstitution ability and will hopefully be of future clinical use in the treatment of hematologic diseases.


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