Regulation of hematopoietic stem cells by the niche

The quiescent state in the cell cycle is thought to be indispensable for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as niches, is critical for maintaining the stem cell properties of HSCs, including cell adhesion, survival, and cell division. Hematopoietic stem cells balance quiescence and cell division in the stem cell niche and also maintain the potential for long-term hematopoiesis. We have recently reported that HSCs expressing the receptor tyrosine kinase Tie2 are in the G0 phase and anti-apoptotic, and comprise a side-population (SP) of HSCs, which contacts osteoblasts (OBs), the source of the angiopoietin-1 (Ang-1) ligand for Tie2 in the bone marrow (BM) niche. Tie2/Ang-1 signaling occurs in interactions between HSCs and niche cells. The interaction of Tie2 with Ang-1 in vitro induces tight adhesion of HSCs to stromal cells and is sufficient to maintain the long-term blood-repopulating (LTR) activity of HSCs in vivo by preventing cell division. In addition, Ang-1 enhances the ability of HSCs to become quiescent and induces their adhesion to the bone surface in vivo, resulting in protection of the HSC compartment from stresses suppressing hematopoiesis. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in the adult BM niche. Ang-1 produced by OBs activates Tie2 on HSCs and promotes tight adhesion of HSCs to the niche, resulting in quiescence and enhanced survival of HSCs.     

Rho GTPase Cdc42 coordinates hematopoietic stem cell quiescence and niche interaction in the bone marrow

Adult hematopoietic stem cells (HSCs) exist in a relatively quiescent state in the bone marrow (BM) microenvironment to fulfill long-term self-renewal and multilineage differentiation functions, an event that is tightly regulated by extrinsic and intrinsic cues. However, the mechanism coordinating the quiescent state of HSCs and their retention in the BM microenvironment remains poorly understood. In a conditional-knockout mouse model, we show that Cdc42(-/-) HSCs enter the active cell cycle, resulting in significantly increased number and frequency of the stem/progenitor cells in the BM. Cdc42 deficiency also causes impaired adhesion, homing, lodging, and retention of HSCs, leading to massive egress of HSCs from BM to distal organs and peripheral blood and to an engraftment failure. These effects are intrinsic to the HSCs and are associated with deregulated c-Myc, p21(Cip1), beta1-integrin, and N-cadherin expressions and defective actin organization. Thus, Cdc42 is a critical coordinator of HSC quiescence maintenance and interaction with the BM niche.     

JAM-B regulates maintenance of hematopoietic stem cells in the bone marrow

In adult mammals, hematopoietic stem cells (HSCs) reside in the bone marrow (BM) and are maintained in a quiescent and undifferentiated state through adhesive interactions with specialized microenvironmental niches. Although junctional adhesion molecule-C (JAM-C) is expressed by HSCs, its function in adult hematopoiesis remains elusive. Here, we show that HSCs adhere to JAM-B expressed by BM stromal cells in a JAM-C dependent manner. The interaction regulates the interplay between HSCs and BM stromal cells as illustrated by the decreased pool of quiescent HSCs observed in jam-b deficient mice. We further show that this is probably because of alterations of BM stromal compartments and changes in SDF-1α BM content in jam-b(-/-) mice, suggesting that JAM-B is an active player in the maintenance of the BM stromal microenvironment.     

TGF-beta signaling-deficient hematopoietic stem cells have normal self-renewal and regenerative ability in vivo despite increased proliferative capacity in vitro

Studies in vitro implicate transforming growth factor beta (TGF-beta) as a key regulator of hematopoiesis with potent inhibitory effects on progenitor and stem cell proliferation. In vivo studies have been hampered by early lethality of knock-out mice for TGF-beta isoforms and the receptors. To directly assess the role of TGF-beta signaling for hematopoiesis and hematopoietic stem cell (HSC) function in vivo, we generated a conditional knock-out model in which a disruption of the TGF-beta type I receptor (T beta RI) gene was induced in adult mice. HSCs from induced mice showed increased proliferation recruitment when cultured as single cells under low stimulatory conditions in vitro, consistent with an inhibitory role of TGF-beta in HSC proliferation. However, induced T beta RI null mice show normal in vivo hematopoiesis with normal numbers and differentiation ability of hematopoietic progenitor cells. Furthermore HSCs from T beta RI null mice exhibit a normal cell cycle distribution and do not differ in their ability long term to repopulate primary and secondary recipient mice following bone marrow transplantation. These findings challenge the classical view that TGF-beta is an essential negative regulator of hematopoietic stem cells under physiologic conditions in vivo.     

Both alleles of PSF1 are required for maintenance of pool size of immature hematopoietic cells and acute bone marrow regeneration

Hematopoietic stem cells (HSCs) have a very low rate of cell division in the steady state; however, under conditions of hematopoietic stress, these cells can begin to proliferate at high rates, differentiate into mature hematopoietic cells, and rapidly reconstitute ablated bone marrow (BM). Previously, we isolated a novel evolutionarily conserved DNA replication factor, PSF1 (partner of SLD5-1), from an HSC-specific cDNA library. In the steady state, PSF1 is expressed predominantly in CD34(+)KSL (c-kit(+)/Sca-1(+)/Lineage(-)) cells and progenitors, whereas high levels of PSF1 expression are induced in KSL cells after BM ablation. In 1-year-old PSF1(+/-) mice, the pool size of stem cells and progenitors is decreased. Whereas young PSF1(+/-) mutant mice develop normally, are fertile, and have no obvious differences in hematopoiesis in the steady state compared with wild-type mice, intravenous injection of 5-fluorouracil (5-FU) is lethal in PSF1(+/-) mice, resulting from a delay in induction of HSC proliferation during ablated BM reconstitution. Overexpression studies revealed that PSF1 regulates molecular stability of other GINS components, including SLD5, PSF2, and PSF3. Our data indicate that PSF1 is required for acute proliferation of HSCs in the BM of mice.     

Necdin, a p53 target gene, regulates the quiescence and response to genotoxic stress of hematopoietic stem/progenitor cells

We recently defined a critical role for p53 in regulating the quiescence of adult hematopoietic stem cells (HSCs) and identified necdin as a candidate p53 target gene. Necdin is a growth-suppressing protein and the gene encoding it is one of several that are deleted in patients with Prader-Willi syndrome. To define the intrinsic role of necdin in adult hematopoiesis, in the present study, we transplanted necdin-null fetal liver cells into lethally irradiated recipients. We show that necdin-null adult HSCs are less quiescent and more proliferative than normal HSCs, demonstrating the similar role of necdin and p53 in promoting HSC quiescence during steady-state conditions. However, wild-type recipients repopulated with necdin-null hematopoietic stem/progenitor cells show enhanced sensitivity to irradiation and chemotherapy, with increased p53-dependent apoptosis, myelosuppression, and mortality. Necdin controls the HSC response to genotoxic stress via both cell-cycle-dependent and cell-cycle-independent mechanisms, with the latter occurring in a Gas2L3-dependent manner. We conclude that necdin functions as a molecular switch in adult hematopoiesis, acting in a p53-like manner to promote HSC quiescence in the steady state, but suppressing p53-dependent apoptosis in response to genotoxic stress.     

Quiescence regulators for hematopoietic stem cell

Hematopoietic stem cell (HSC) either stays in quiescence or proliferates toward differentiation for the production of mature blood cells, or toward self-renewal for giving rise to itself. In order to both maintain a supply of mature blood cells and not exhaust HSCs throughout the lifetime of an individual, under steady state, most HSCs remain quiescent and only a small number enter the cell cycle. Quiescence of HSCs is not only critical for protecting the stem cell compartment and sustaining stem cell pools over long periods, but it is also critical for protecting stem cells by minimizing their accumulation of replication-associated mutations. The balance between quiescence and proliferation is tightly controlled by both HSC-intrinsic and -extrinsic mechanisms. In recent years, through reductionistic strategies, a wide variety of molecules or pathways critical for HSC quiescence regulation have been identified. This regulation network involves both positive and negative regulators. Understanding quiescence regulation in HSC is of great importance not only for understanding the physiological foundation of HSCs, but also for understanding the pathophysiological origins of many related disorders. In this article, I will briefly review the current advance in the quiescence regulators for the HSCs.     

Influences of vascular niches on hematopoietic stem cell fate

The fate decision of hematopoietic stem cells (HSCs), quiescence, proliferation or differentiation, is uniquely determined by functionally specialized microenvironments defined as the HSC niches. However, whether quiescence and proliferation of HSCs are regulated by spatially distinct niches is unclear. Although various candidate stromal cells have been identified as potential niche cells, the spatial localization of quiescent HSCs in the bone marrow remains controversial. In our recent study, we have established whole-mount confocal immunofluorescence techniques, which allow us to precisely assess the localization of HSCs and their relationships with stromal structures. Furthermore, we have assessed the significance of these associations using a computational simulation. These novel analyses have revealed that quiescent HSCs are specifically associated with small caliber arterioles, which are predominantly distributed in the endosteal bone marrow while the associations with sinusoidal vessels or osteoblasts are not significant. Physical ablation of the arteriolar niche causes the shift of HSC localization to sinusoidal niches, where HSCs are switched into non-quiescent status. This new imaging analyses together with previous studies suggest the presence of spatially distinct vascular niches for quiescent and non-quiescent (proliferating) HSCs in the bone marrow.     

The role of Skp2 in hematopoietic stem cell quiescence, pool size, and self-renewal

Although the maintenance of HSC quiescence and self-renewal are critical for controlling stem cell pool and transplantation efficiency, the mechanisms by which they are regulated remain largely unknown. Understanding the factors controlling these processes may have important therapeutic potential for BM failure and cancers. Here, we show that Skp2, a component of the Skp2 SCF complex, is an important regulator for HSC quiescence, frequency, and self-renewal capability. Skp2 deficiency displays a marked enhancement of HSC populations through promoting cell cycle entry independently of its role on apoptosis. Surprisingly, Skp2 deficiency in HSCs reduces quiescence and displays increased HSC cycling and proliferation. Importantly, loss of Skp2 not only increases HSC populations and long-term reconstitution ability but also rescues the defect in long-term reconstitution ability of HSCs on PTEN inactivation. Mechanistically, we show that Skp2 deficiency induces Cyclin D1 gene expression, which contributes to an increase in HSC cycling. Finally, we demonstrate that Skp2 deficiency enhances sensitivity of Lin(-) Sca-1(+) c-kit(+) cells and leukemia cells to chemotherapy agents. Our findings show that Skp2 is a novel regulator for HSC quiescence and self-renewal and that targeting Skp2 may have therapeutic implications for BM transplantation and leukemia stem cell treatment.     

Prdm16 is a physiologic regulator of hematopoietic stem cells

Fetal liver and adult bone marrow hematopoietic stem cells (HSCs) renew or differentiate into committed progenitors to generate all blood cells. PRDM16 is involved in human leukemic translocations and is expressed highly in some karyotypically normal acute myeloblastic leukemias. As many genes involved in leukemogenic fusions play a role in normal hematopoiesis, we analyzed the role of Prdm16 in the biology of HSCs using Prdm16-deficient mice. We show here that, within the hematopoietic system, Prdm16 is expressed very selectively in the earliest stem and progenitor compartments, and, consistent with this expression pattern, is critical for the establishment and maintenance of the HSC pool during development and after transplantation. Prdm16 deletion enhances apoptosis and cycling of HSCs. Expression analysis revealed that Prdm16 regulates a remarkable number of genes that, based on knockout models, both enhance and suppress HSC function, and affect quiescence, cell cycling, renewal, differentiation, and apoptosis to various extents. These data suggest that Prdm16 may be a critical node in a network that contains negative and positive feedback loops and integrates HSC renewal, quiescence, apoptosis, and differentiation.     

CD48 on hematopoietic progenitors regulates stem cells and suppresses tumor formation

The proliferation and differentiation of adult stem cells is balanced to ensure adequate generation of differentiated cells, stem cell homeostasis, and guard against malignant transformation. CD48 is broadly expressed on hematopoietic cells but excluded from quiescent long-term murine HSCs. Through its interactions with CD244 on progenitor cells, it influences HSC function by altering the BM cytokine milieu, particularly IFNγ. In CD48-null mice, the resultant misregulation of cytokine signaling produces a more quiescent HSC, a disproportionate number of short-term progenitors, and hyperactivation of Pak1, leading to hematologic malignancies similar to those found in patients with X-linked lymphoproliferative disease. CD48 plays a vital role as an environmental sensor for regulating HSC and progenitor cell numbers and inhibiting tumor development.     

Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34(+) cells transplanted into conditioned NOD/SCID recipients

Differences in engraftment potential of hematopoietic stem cells (HSCs) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) and may be influenced by the rate of entry of BM-homed HSCs into cell cycle. Alternatively, preferential apoptosis of cycling cells may contribute to their low engraftment potential. This study examined homing, cell cycle progression, and survival of human hematopoietic cells transplanted into nonobese diabetic severe combined immunodeficient (NOD/SCID) recipients. At 40 hours after transplantation (AT), only 1% of CD34(+) cells, or their G(0) (G(0)CD34(+)) or G(1) (G(1)CD34(+)) subfractions, was detected in the BM of recipient mice, suggesting that homing of engrafting cells to the BM was not specific. BM of NOD/SCID mice receiving grafts containing approximately 50% CD34(+) cells harbored similar numbers of CD34(+) and CD34(-) cells, indicating that CD34(+) cells did not preferentially traffic to the BM. Although more than 64% of human hematopoietic cells cycled in culture at 40 hours, more than 92% of cells recovered from NOD/SCID marrow were quiescent. Interestingly, more apoptotic human cells were detected at 40 hours AT in the BM of mice that received xenografts of expanded cells in S/G(2)+M than in recipients of G(0)/G(1) cells (34.6% +/- 5.9% and 17.1% +/- 6.3%, respectively; P <.01). These results suggest that active proliferation inhibition in the BM of irradiated recipients maintains mitotic quiescence of transplanted HSCs early AT and may trigger apoptosis of cycling cells. These data also illustrate that trafficking of transplanted cells to the BM is not selective, but lodgment of BM-homed cells may be specific.     

CD133 is a modifier of hematopoietic progenitor frequencies but is dispensable for the maintenance of mouse hematopoietic stem cells

Pentatransmembrane glycoprotein prominin-1 (CD133) is expressed at the cell surface of multiple somatic stem cells, and it is widely used as a cell surface marker for the isolation and characterization of human hematopoietic stem cells (HSCs) and cancer stem cells. CD133 has been linked on a cell biological basis to stem cell-fate decisions in human HSCs and emerges as an important physiological regulator of stem cell maintenance and expansion. Its expression and physiological relevance in the murine hematopoietic system is nevertheless elusive. We show here that CD133 is expressed by bone marrow-resident murine HSCs and myeloid precursor cells with the developmental propensity to give rise to granulocytes and monocytes. However, CD133 is dispensable for the pool size and function of HSCs during steady-state hematopoiesis and after transplantation, demonstrating a substantial species difference between mouse and man. Blood cell numbers in the periphery are normal; however, CD133 appears to be a modifier for the development of growth-factor responsive myeloerythroid precursor cells in the bone marrow under steady state and mature red blood cells after hematopoietic stress. Taken together, these studies show that CD133 is not a critical regulator of hematopoietic stem cell function in mouse but that it modifies frequencies of growth-factor responsive hematopoietic progenitor cells during steady state and after myelotoxic stress in vivo.     

Non-side-population hematopoietic stem cells in mouse bone marrow

Most hematopoietic stem cells (HSCs) are assumed to reside in the so-called side population (SP) in adult mouse bone marrow (BM). We report the coexistence of non-SP HSCs that do not significantly differ from SP HSCs in numbers, capacities, and cell-cycle states. When stained with Hoechst 33342 dye, the CD34(-/low) c-Kit(+)Sca-1(+)lineage marker(-) (CD34(-)KSL) cell population, highly enriched in mouse HSCs, was almost equally divided into the SP and the main population (MP) that represents non-SP cells. Competitive repopulation assays with single or 30 SP- or MP-CD34(-)KSL cells found similar degrees of repopulating activity and frequencies of repopulating cells for these populations. Secondary transplantation detected self-renewal capacity in both populations. SP analysis of BM cells from primary recipient mice suggested that the SP and MP phenotypes are interconvertible. Cell-cycle analyses revealed that CD34(-)KSL cells were in a quiescent state and showed uniform cell-cycle kinetics, regardless of whether they were in the SP or MP. Bcrp-1 expression was similarly detected in SP- and MP-CD34(-)KSL cells, suggesting that the SP phenotype is regulated not only by Bcrp-1, but also by other factors. The SP phenotype does not specify all HSCs; its identity with stem cell function thus is unlikely.     

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.     

Accelerated recovery of peripheral blood cell counts in mice transplanted with in vitro cytokine-expanded hematopoietic progenitors.

Interleukin 1 (IL-1) and interleukin 3 (IL-3) act synergistically in stimulating the growth of primitive hematopoietic progenitors. Murine bone marrow (BM) harvested 24 h after 5-fluorouracil (5-FU) administration (d1 5-FU BM) was stimulated with IL-1 and IL-3 to expand its progenitor pool during 7 days of suspension culture (delta-culture), and this in vitro expanded BM was compared to fresh d1 5-FU BM in its ability to reconstitute lethally irradiated or high-dose 5-FU-treated hosts. Transplantation with expanded delta-culture BM was found to dramatically shorten the period of cytopenia following lethal irradiation as compared to animals receiving d1 5-FU BM. Recipients of delta-cultured BM demonstrated accelerated recoveries of peripheral blood leukocytes, neutrophils, platelets, and erythrocytes. Furthermore, expansion of BM in vitro reduced the number of BM cells required for engraftment following lethal irradiation. Treatment of lethally irradiated mice with IL-1 and granulocyte colony-stimulating factor (G-CSF) following transplantation with delta-cultured BM or d1 5-FU BM further improved the recovery of neutrophils in these hosts. In conjunction with G-CSF post-transplantation cytokine therapy, high-dose 5-FU-treated mice transplanted with delta-cultured BM also demonstrated improved recovery kinetics of neutrophils and erythrocytes. Five and 10 weeks after BM transplantation, a decrease in the proliferative capacity of the earliest hematopoietic progenitors, detected in assays of primary and delta-culture generated-secondary high proliferative potential colony-forming cells (HPP-CFC), was found in all transplanted mice following a chemotherapy challenge with 5-FU. However, this impairment in the early progenitor/stem cell pool was not noticeably worsened by the expansion of BM in delta-cultures. The decrease in host hematopoietic proliferative potential associated with transplantation of limiting numbers of BM cells was not reversed over the 10 weeks of this study. The expansion of BM progenitor cells without loss of long-term proliferative potential may be of clinical importance in the fields of BM transplantation and gene therapy.