Autonomous behavior of hematopoietic stem cells

Mechanisms that affect the function of primitive hematopoietic stem cells with long-term proliferative potential remain largely unknown. Here we assessed whether properties of stem cells are cell-extrinsically or cell-autonomously regulated.We developed a model in which two genetically and phenotypically distinct stem cell populations coexist in a single animal. Chimeric mice were produced by transplanting irradiated B6D2F1 (BDF1) recipients with mixtures of DBA/2 (D2) and C57BL/6 (B6) day-14 fetal liver cells.We determined the mobilization potential, proliferation, and frequency of D2 and B6 stem and progenitor cells in animals with chimeric hematopoiesis. After granulocyte colony-stimulating factor (G-CSF) administration, peripheral blood D2 colony-forming units granulocyte-macrophage were fourfold to eightfold more numerous than B6 progenitors. We determined that D2 and B6 progenitors maintained their genotype-specific cycling activity in BDF1 recipients. Chimeric marrow was harvested and D2 and B6 cell populations were separated by flow cytometry. Cobblestone area-forming cell (CAFC) analysis of sorted marrow showed that the number of late appearing CAFC subsets within the D2 cell population was approximately threefold higher than within the B6 fraction. We performed secondary transplantation using unfractionated chimeric marrow, which was given in limiting doses to lethally irradiated BDF1 recipients. Comparison of the proportion of animals possessing D2 and/or B6 leukocytes 5 months after transplant revealed that the frequency of D2 LTRA was approximately 10-fold higher than B6 LTRA numbers.Our data demonstrate that genetically distinct stem cell populations, coexisting in individual animals, independently maintain their parental phenotypes, indicating that stem cell properties are predominantly regulated cell-autonomously.     

Kinetics and symmetry of divisions of hematopoietic stem cells

To fulfill the dual abilities to self-renew and to differentiate into cells of multiple lineages, stem cells must undergo, at some stage, asymmetric divisions to generate cells to sustain the stem cell pool as well as the various progeny cells of the distinct lineages. A central question in developmental biology is how a single cell can divide to produce two progeny cells that adopt different fates. Different daughter cells can theoretically arise by uneven distribution of determinants upon cell division, i.e., due to intrinsic factors, or become different upon subsequent exposure to environmental signals, i.e., due to extrinsic factors. Recent advances in the understanding of stem cell biology in Drosophila and murine models have served as a model for hematopoietic stem cell (HSC) development. Provided with advances in molecular and cellular biology, we have gained insight into the mechanisms governing self-renewing asymmetric divisions of primitive HSC. Direct contact with cellular determinants in the niche has been shown to play an essential role in the balance between self-renewing asymmetric division versus differentiation. Identification of the molecular interactions between stem cells and their niche will lead to an understanding of the mechanisms controlling the long-term destiny of stem cells. Ultimately, molecular signals triggered by adhesion and junction complexes are probably responsible for the specific adoption of differentiation pathways.     

Stromal pleiotrophin regulates repopulation behavior of hematopoietic stem cells

Pleiotrophin (Ptn) is strongly expressed by stromal cells which maintain HSCs. However, in vivo, Ptn deficiency does not alter steady-state hematopoiesis. However, knockdown of Ptn (Ptn(KD)) in stromal cells increases production of hematopoietic progenitors as well as HSC activity in cocultures, suggesting that Ptn may have a role in HSC activation. Indeed, transplantations of wild-type (Ptn(+/+)) HSCs into Ptn(-/-) mice show increased donor cell production in serial transplantations and dominant myeloid regeneration caused by Ptn-dependent regulation of HSC repopulation behavior. This regulation of Lin(-)Kit(+)Sca1(+) function is associated with increased proliferation and, on a molecular level, with up-regulated expression of cyclin D1 (Ccnd1) and C/EBPα (Cepba), but reduced of PPARγ. The known HSC regulator β-catenin is, however, not altered in the absence of Ptn. In conclusion, our results point to different Ptn-mediated regulatory mechanisms in normal hemostasis and in hematopoietic regeneration and in maintaining the balance of myeloid and lymphoid regeneration. Moreover, our results support the idea that microenvironmental Ptn regulates hematopoietic regeneration through β-catenin-independent regulation of Ccnd1 and Cebpa.     

High-resolution video monitoring of hematopoietic stem cells cultured in single-cell arrays identifies new features of self-renewal

To search for new indicators of self-renewing hematopoietic stem cells (HSCs), highly purified populations were isolated from adult mouse marrow, micromanipulated into a specially designed microscopic array, and cultured for 4 days in 300 ng/ml Steel factor, 20 ng/ml IL-11, and 1 ng/ml flt3-ligand. During this period, each cell and its progeny were imaged at 3-min intervals by using digital time-lapse photography. Individual clones were then harvested and assayed for HSCs in mice by using a 4-month multilineage repopulation endpoint (>1% contribution to lymphoid and myeloid lineages). In a first experiment, 6 of 14 initial cells (43%) and 17 of 61 clones (28%) had HSC activity, demonstrating that HSC self-renewal divisions had occurred in vitro. Characteristics associated with HSC activity included longer cell-cycle times and the absence of uropodia on a majority of cells within the clone during the final 12 h of culture. Combining these criteria maximized the distinction of clones with HSC activity from those without and identified a subset of 27 of the 61 clones. These 27 clones included all 17 clones that had HSC activity; a detection efficiency of 63% (2.26 times more frequently than in the original group). The utility of these characteristics for discriminating HSC-containing clones was confirmed in two independent experiments where all HSC-containing clones were identified at a similar 2- to 3-fold-greater efficiency. These studies illustrate the potential of this monitoring system to detect new features of proliferating HSCs that are predictive of self-renewal divisions.     

Neonatal W-mutant mice are favorable hosts for tracking development of marked hematopoietic stem cells

Neonatal unirradiated mice of W-mutant genotypes, with a hematopoietic stem cell defect and anemia, were injected i.v. with normal fetal liver hematopoietic cells. Efficient, long-term engraftment occurred as a result of the competitive advantage to the donor stem cells. The frequency of engraftment and rate of repopulation characteristically diminish in the series W/Wv, Wf/Wf, and Wv/+, in which the severity of the endogenous defect is progressively less. H-2 compatibility is required in the inbred strain combinations examined; other histocompatibility loci play a minor role in some strain combinations. Engraftment is due to self-renewing hematopoietic stem cells ancestral to myeloid and lymphoid lineages. The more mildly defective mutants display much greater variability in the kinetics of repopulation--a result consistent with seeding by single, or very few, stem cells that form developing clones. Engraftment efficiency is reduced by prolonged culture of fetal liver cells during experimental infection by recombinant retroviruses; nevertheless, after 24 h in vitro to achieve retroviral marking, stem cells retain their ability to repopulate and develop in W/Wv neonates.     

Bone and the hematopoietic niche: a tale of two stem cells

The revived interest in (hematopoietic) stem cell (HSC) niches has highlighted the role of multiple cellular players found in the bone environment. Initially focused on the role of osteoblasts and sinusoid endothelial cells, the quest for HSC niche cells has recently focused on a unique role for osteoprogenitor cells (skeletal stem cells, mesenchymal stem cells). Strongly validated by observations of HSC dysregulation dictated by the dysregulation of osteoprogenitors, the role of osteoprogenitors in the HSC niche integrates data from different studies into a unified view. As preosteoblastic, periendothelial cells residing at the sinusoid wall, skeletal progenitors reconcile the notions of "osteoblastic" and "sinusoidal" niches with one another. In addition, they bring into focus the cross-regulation of skeletal and hematopoietic physiology as rooted into the interplay of two stem cells (hematopoietic and skeletal) sharing a single niche. As direct regulators of hematopoietic space formation, sinusoid development, and hematopoietic function(s), as well as direct progenitors of positive and negative regulators of HSCs such as osteoblasts and adipocytes, skeletal progenitors have emerged as pivotal organizers of a complex, highly plastic niche. This development seems to represents an evolutionary advance over the deterministic stem cell niches found in archetypal invertebrate systems.     

Screening test for hematopoietic stem cells in umbilical cord blood with the SE-9000 automated hematology analyzer

The immature information IMI channel on an SE-9000 automated hematology analyzer was used for detection of stem cells in cord blood and the results were compared with other standard methods, including flow cytometry analysis and progenitor assays. After the removal of red blood cells, the IMI count in cord blood samples significantly correlated with the number of CD34+ cells (r = 0.810), CFU-GM (r = 0.606) and BFU-E (r = 0.961). The cell count in hematopoietic progenitor cell (HPC) area also showed a correlation with CD34+ cell number (r = 0.722), but to a lesser extent than the IMI count; no significant correlations were observed with the numbers of progenitor cells. Cord blood contained higher fractions of CD34+/CD38- and CD34+/CD117+ cells representative of immature subclasses of progenitor cells. This might explain the difference in the HPC module results for the cord blood and peripheral blood stem cell samples. A higher coefficient of correlation was obtained with leukocyte suspensions than with whole blood samples. These results demonstrated the usefulness of IMI-positive cell measurement as a stem cell screening test for cord blood banking.     

Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells.

Clonogenic multipotent mouse hematopoietic stem cells (HSCs) and progenitor cells are contained within the c-kit(+) (K) lineage(-/lo) (L) Sca-1(+) (S) population of hematopoietic cells; long-term (LT) and short-term (ST) HSCs are Thy-1.1(lo). c-kit is a member of the receptor tyrosine kinase family, a class of receptors that are important in the proliferation and differentiation of hematopoietic cells. To establish whether the Flk-2/Flt3 receptor tyrosine kinase was expressed on the most primitive LT-HSCs, we sorted highly purified multipotent stem and progenitor cells on the basis of Flk-2 surface expression and used them in competitive reconstitution assays. Low numbers of Flk-2(-) HSCs gave rise to long-term multilineage reconstitution in the majority of recipients, whereas the transfer of Flk-2(+) multipotent cells resulted in mostly short-term multilineage reconstitution. The KLS subset of adult mouse bone marrow was analyzed for Flk-2 and Thy-1.1 expression. Three phenotypically and functionally distinct populations were isolated: Thy(lo) Flk-2(-) (LT-HSCs), Thy(lo) Flk-2(+) (ST-HSCs), and Thy(-) Flk-2(+) multipotent progenitors. The loss of Thy-1.1 and gain of Flk-2 expression marks the loss of self-renewal in HSC maturation. The addition of Flk-2 antibody to the lineage mix allows direct isolation of LT-HSC from adult bone marrow as c-kit(+) lin(-) Sca-1(+) Flk-2(-) from many strains of mice. Fetal liver HSCs are contained within Flk-2(-) and Flk-2(+) KTLS cells.     

Aging of the hematopoietic stem cells niche

Homeostasis of the hematopoietic system has its roots in the maintenance of hematopoietic stem cells (HSCs) in the bone marrow (BM). HSCs change both phenotypically and functionally with physiological age. The alterations noted in aged HSCs are thought to be a consequence of both cell-intrinsic and extrinsic changes. We review here the age-related changes that the BM microenvironment exerts on HSCs.     

Multipotential hematopoietic stem cells in the bone marrow

It has been generally held that human hematopoietic stem cells are lineage-negative CD34⁺ CD38^?. However, murine hematopoietic stem cells were reported to be CD34^?. We have characterized the surface phenotypes of murine hematopoietic stem cells by using a murine transplantation model. Our studies revealed that the majority of the stem cells in normal adult mice are CD34^? while a minority (15%–20%) being CD34⁺. Our studies also revealed that stem cells that are activated by injection of 5-fluorouracil in vivo, exposure to cytokines in vitro, or mobilization by G-CSF are CD34⁺ and that CD34 expression is reversible. It has been reported that fetal murine hematopoietic stem cells are CD34⁺. Our studies revealed that stem cells of juvenile mice are CD34+ and that the developmental change from CD34⁺ to CD34^? state takes place between 7 and 10 weeks of age. In adult mice, expression of CD38 by steady-state and activated stem cells was completely reciprocal of CD34 expression. Activated stem cells and the minority population of the stem cells in the normal mice are CD34⁺ CD38^?. In contrast, the majority of stem cells in normal adult mice are CD34- CD38+. Recently, we studied CD38 expression by stem cells of neonatal and juvenile mice. Stem cells of newborn mice are CD38^?. About half of the stem cells of 5-week-old mice are CD38⁺. Finally, our studies indicated that some of the CD34⁺ stem cells in the bone marrow of normal adult mice express lineage markers such as Mac-1 and CD4. These studies in a murine model clearly documented that expression of both CD34 and CD38 by stem cells is under developmental control and may be subject to changes induced by activation of the stem cells. In order to test whether or not these principles apply to human stem cells we tested surface phenotypes of human stem cells using two xenotransplantation techniques. Studies based on human/sheep xenograft model indicated that a significant portion of adult human long-term engrafting cells are CD34^?. Similar to mouse stem cells expression of CD34 by human stem cells was reversible. Studies based on our newborn NOD/SCID/β-microglobulin^null mice indicated that human cord blood stem cells are CD34⁺ CD38^?. These results appear to support the validity of studies of murine stem cells to provide insight into human stem cells.     

Behavior of hematopoietic stem cells in a large animal.

To study the behavior of hematopoietic stem cells in vivo, we transplanted glucose-6-phosphate dehydrogenase (G6PD) heterozygous (female Safari) cats with small amounts of autologous marrow. The G6PD phenotypes of erythroid burst-forming units and granulocyte/macrophage colony-forming units were repeatedly assayed for 3.5-6 years after transplantation to track contributions of stem cell clones to the progenitor cell compartment. Two phases of stem cell kinetics were observed, which were similar to the pattern reported in comparable murine studies. Initially there were significant fluctuations in contributions of stem cell clones. Later clonal contributions to hematopoiesis stabilized. The initial phase of clonal disequilibrium, however, extended for 1-4.5 years (and not 2-6 months as seen in murine experiments). After this subsided, all progenitor cells from some animals expressed a single parental G6PD phenotype, suggesting that blood cell production could be stably maintained by the progeny of one (or a few) cells. As the hematopoietic demand of a cat (i.e., number of blood cells produced per lifetime) is over 600 times that of a mouse, this provides evidence that an individual hematopoietic stem cell has a vast self-renewal and/or proliferative capacity. The long phase of clonal instability may reflect the time required for stem cells to replicate sufficiently to reconstitute a large stem cell reserve.     

The contribution of hematopoietic stem cells to beta-cell replacement

Hemopoietic stem cells (HSCs) are commonly used for curing malignant and nonmalignant hematopoiesis disorders. In recent years, HSC potential giving rise to multilineage progeny has been reported. This issue, together with their availability and number, has made them ideal candidates for β-cell replacement in diabetic patients. HSC capacity to differentiate to insulin-producing cells has been at the center of debate for the past 5 years and it now seems that their role could more likely be that of helper cells able to facilitate survival or stimulate proliferation of endogenous β cells. In addition, clinical studies are ongoing about the possible use of HSCs to stop autoimmune destruction at the onset of diabetes or to induce tolerance through microchimerism in pancreatic islet transplantation.     

Towards hematopoietic reconstitution from embryonic stem cells: a sanguine future

PURPOSE OF REVIEW: To review recent progress towards the derivation of hematopoietic stem cells (HSCs) and blood lineages from embryonic stem cells (ESCs), and to highlight the hurdles that must be overcome in order to move the field closer to a clinical application. RECENT FINDINGS: Hematopoietic repopulating cells, red blood cells, and T cells have recently been derived from both murine and human ESCs. Although these results are encouraging, several outstanding issues remain to be addressed by the field before realizing clinical applicability: the phenotype of the ESC-derived HSC must be characterized, methods to purge residual teratoma-forming cells from differentiated populations must be established, and in-vivo models of human HSC function must be optimized to better assess the functionality of putative human ESC-derived HSCs. In addition, embryonic stem-cell derived progeny often represent primitive embryonic hematopoietic cells, rather than their definitive adult counterparts; this critical issue must also be addressed. SUMMARY: The literature firmly establishes that it is possible to isolate HSCs and certain mature blood lineages from both mouse and human ESCs. Although several issues remain to be addressed, these data demonstrate the value of ESCs as a potential source of transplantable HSCs.     

Role of mesenchymal stem cells in hematopoietic stem cell transplantation

Within the bone marrow stroma are multipotential cells which are capable of differentiation into a number of mesenchymal cell lineages. These cells, termed mesenchymal stem cells, have recently been identified and characterized in humans. Many studies indicate that the bone marrow stroma is damaged following bone marrow transplantation. Since the marrow stroma is critical for the maintenance of hematopoiesis, its ability to support hematopoiesis following stem cell transplantation may be impaired. Animal models suggest that the transplantation of healthy stromal elements, including mesenchymal stem cells, may enhance the ability of the bone marrow microenvironment to support hematopoiesis after stem cell transplantation. Here the authors review recent data that suggest that mesenchymal stem cells may possess therapeutic value not only for the repair of damaged mesenchymal tissues following hematopoietic stem cell transplantation, but also as potential vectors for the delivery of corrective genes.     

Characterization of a novel hematopoietic marker expressed from early embryonic hematopoietic stem cells to adult mature lineages

A novel membrane protein has been identified in the course of screening for differentially expressed cDNAs in human embryonic hematopoietic sites. This 37- to 38-kDa molecule, designated KLIP-1 (killer lineage protein), consisting of 350 amino acids and containing five transmembrane domains, is encoded by the 5093-bp KLIP-1 gene, composed of nine exons and located on chromosome 6 (6p21.1-6p21.2). We found the KLIP-1 protein to be expressed by nucleated hematopoietic cells, from early embryonic hematopoietic stem cells through mature adult blood lymphoid lineages, either as membrane or as cytoplasmic molecules. In day-30/32 human embryo sections, KLIP-1 protein expression is restricted to circulating hematopoietic cells at hematopoiesis sites. Membrane KLIP-1 is expressed by fetal and adult GP-A(+) erythroblasts, the fetal liver CD34(+) subset, fetal spleen, and adult bone marrow CD56(+) NK and CD19(+) B cells. Among mature blood cells, surface KLIP-1 expression is restricted to CD56(+) NK cells, indicating KLIP-1 to be a novel marker of this population. Altogether, these results indicate that membrane export of KLIP-1 antigen is developmentally and ontogenetically regulated. The high degree of conservation of the KLIP-1 protein sequence among mammals strongly suggests that it plays an important role during hematopoiesis and may exercise similar functions in human and mouse blood cells. The KLIP-1 molecule may therefore constitute a powerful tool for improving knowledge of both human hematopoiesis and NK cell ontogeny and immune functions.