Synergistic use of adult and embryonic stem cells to study human hematopoiesis

Embryonic stem cells (ESCs) and adult stem cells both provide important resources to define the mechanisms of hematopoietic cell development. To date, studies that utilize hematopoietic stem cells (HSCs) isolated from sites such as bone marrow or umbilical cord blood have been the primary means to identify molecular and phenotypic characteristics of blood cell populations able to mediate long-term hematopoietic engraftment. Although these HSCs are very useful clinically, they are difficult to expand in culture. Now, basic research on human ESCs provides opportunities for novel investigations into the mechanisms of HSC self-renewal. Eventually, the long history of basic and clinical research with adult hematopoietic cell transplantation could translate to establish human ESCs as a suitable alternative starting cell source for clinical hematopoietic reconstitution.     

Prostaglandin E2 enhances human cord blood stem cell xenotransplants and shows long-term safety in preclinical nonhuman primate transplant models

Hematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable; however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2 treatment by using human and nonhuman primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in?vitro and enhanced engraftment of unfractionated and CD34(+) hCB after xenotransplantation. In nonhuman primate autologous transplantation, dmPGE2-treated CD34(+) MPBSCs showed stable multilineage engraftment over 1 year postinfusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and nonhuman primates and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial.     

Optimal protocols of hematopoietic stem cell expansion in vitro

Hematopoietic stem cells (HSCs) effectively and continuously replenish the full range of blood-cell populations. Bone-marrow and umbilical-cord blood stem-cell transplantation (SCT) restore hematopoiesis when used in various hematological and oncohematological disorders in adults and children. However, wider clinical application of effective SCT-based approaches is limited by the low number of primitive HSCs in the available biospecimens. Development of effective protocols of HSC expansion in vitro is therefore necessary. In this review, the notion of bone marrow hematopoiesis is discussed as a complex cellular system and a comparative analysis of various methods for HSC expansion in vitro is provided. The review is illustrated by our own data supporting application of various feeder-cell types for human HSC expansion in vitro.     

Osteopontin attenuates aging-associated phenotypes of hematopoietic stem cells

Upon aging, hematopoietic stem cells (HSCs) undergo changes in function and structure, including skewing to myeloid lineages, lower reconstitution potential and loss of protein polarity. While stem cell intrinsic mechanisms are known to contribute to HSC aging, little is known on whether age-related changes in the bone marrow niche regulate HSC aging. Upon aging, the expression of osteopontin (OPN) in the murine bone marrow stroma is reduced. Exposure of young HSCs to an OPN knockout niche results in a decrease in engraftment, an increase in long-term HSC frequency and loss of stem cell polarity. Exposure of aged HSCs to thrombin-cleaved OPN attenuates aging of old HSCs, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity and a restored balance of lymphoid and myeloid cells in peripheral blood. Thus, our data suggest a critical role for reduced stroma-derived OPN for HSC aging and identify thrombin-cleaved OPN as a novel niche informed therapeutic approach for ameliorating HSC phenotypes associated with aging.     

Robo4 cooperates with CXCR4 to specify hematopoietic stem cell localization to bone marrow niches

Specific bone marrow (BM) niches are critical for hematopoietic stem cell (HSC) function during both normal hematopoiesis and in stem cell transplantation therapy. We demonstrate that the guidance molecule Robo4 functions to specifically anchor HSCs to BM niches. Robo4-deficient HSCs displayed poor localization to BM niches and drastically reduced long-term reconstitution capability while retaining multilineage potential. Cxcr4, a critical regulator of HSC location, is upregulated in Robo4(-/-) HSCs to compensate for Robo4 loss. Robo4 deletion led to altered HSC mobilization efficiency, revealing that inhibition of both Cxcr4- and Robo4-mediated niche interactions are necessary for efficient HSC mobilization. Surprisingly, we found that WT HSCs express very low levels of Cxcr4 and respond poorly to Cxcr4 manipulation relative to other hematopoietic cells. We conclude that Robo4 cooperates with Cxcr4 to endow HSCs with competitive access to limited stem cell niches, and we propose Robo4 as a therapeutic target in HSC transplantation therapy.     

Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells

Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood production for their lifetime. Appropriate control of HSC self-renewal is crucial for the maintenance of hematopoietic homeostasis. Here we show that activation of p38 MAPK in response to increasing levels of reactive oxygen species (ROS) limits the lifespan of HSCs in vivo. In Atm(-/-) mice, elevation of ROS levels induces HSC-specific phosphorylation of p38 MAPK accompanied by a defect in the maintenance of HSC quiescence. Inhibition of p38 MAPK rescued ROS-induced defects in HSC repopulating capacity and in the maintenance of HSC quiescence, indicating that the ROS-p38 MAPK pathway contributes to exhaustion of the stem cell population. Furthermore, prolonged treatment with an antioxidant or an inhibitor of p38 MAPK extended the lifespan of HSCs from wild-type mice in serial transplantation experiments. These data show that inactivation of p38 MAPK protects HSCs against loss of self-renewal capacity. Our characterization of molecular mechanisms that limit HSC lifespan may lead to beneficial therapies for human disease.     

Proliferative senescence in hematopoietic stem cells during ex-vivo expansion

The good outcome of hematopoietic stem cell (HSC) transplantation is hampered by low doses of CD34+ cell infusion. Transplanted HSCs undergo a replicative stress that causes accelerated senescence due to rapid telomere shortening. The expansion of human cord blood HSCs is instrumental in obtaining a large number of "good quality" cells, in terms of telomere length and telomerase activity compared to adult HSCs.     

Niche-to-niche migration of bone-marrow-derived cells

During ontogenesis, haematopoietic stem cells (HSCs) relocate between extra-embryonic and embryonic compartments. Similarly, site-specific homing of HSCs is ongoing during adulthood. With the expanding knowledge of HSC physiology, a new paradigm emerges in which HSCs and haematopoietic progenitor cells (HPCs) migrate to defined microenvironments within the bone marrow (BM) and to 'activated' or 'inducible' niches elsewhere. Here, we summarize current understanding of HSC niche characteristics, and the physiological and pathological mechanisms that guide HSC homing both within the BM and to distant niches in the periphery, promoting new vessel growth in tumours and ischaemia. Recent observations suggest that features of the HSC niche might also be recapitulated in pre-metastatic sites. Clusters of BM-derived HPCs promote invasion of disseminating cancer cells. Clear clinical benefits can be foreseen by modulating HSCs and their microenvironments, in promoting tissue regeneration, and inhibiting tumourigenesis and cancer metastasis.     

The elements of stem cell self-renewal: a genetic perspective

Every day, the body produces billions of new blood cells. Each of these is derived from a rare cell in the bone marrow called the hematopoietic stem cell (HSC). Because most mature blood cells have a limited lifespan, the ability of HSCs to self-renew and replenish the mature cell compartment is critical to sustaining life. While great progress has been made in isolating HSCs and defining their functional and phenotypic characteristics, the molecular mechanisms that regulate their self-renewal remain a mystery. Over the last few years, alterations in HSC frequency and self-renewal capacity in transgenic and knock-out mice have led to the identification of novel mediators of HSC homeostasis in vivo. These genetically modified mice have revealed that maintenance of survival, proliferation, quiescence, and normal telomere length all contribute to the self-renewal of HSCs. They also highlight the need to test in context of the normal microenvironment the role of signaling molecules such as Notch and Wnt, which have emerged recently as important regulators of HSC self-renewal. The emerging picture these data provide of the regulation of self-renewal in HSCs has provided a better understanding of the basic biology of stem cells and holds promise for designing strategies to improve bone marrow transplantation.     

Smurf2 regulates hematopoietic stem cell self‐renewal and aging

The age‐dependent decline in the self‐renewal capacity of stem cells plays a critical role in aging, but the precise mechanisms underlying this decline are not well understood. By limiting proliferative capacity, senescence is thought to play an important role in age‐dependent decline of stem cell self‐renewal, although direct evidence supporting this hypothesis is largely lacking. We have previously identified the E3 ubiquitin ligase Smurf2 as a critical regulator of senescence. In this study, we found that mice deficient in Smurf2 had an expanded hematopoietic stem cell (HSC) compartment in bone marrow under normal homeostatic conditions, and this expansion was associated with enhanced proliferation and reduced quiescence of HSCs. Surprisingly, increased cycling and reduced quiescence of HSCs in Smurf2‐deficient mice did not lead to premature exhaustion of stem cells. Instead, HSCs in aged Smurf2‐deficient mice had a significantly better repopulating capacity than aged wild‐type HSCs, suggesting that decline in HSC function with age is Smurf2 dependent. Furthermore, Smurf2‐deficient HSCs exhibited elevated long‐term self‐renewal capacity and diminished exhaustion in serial transplantation. As we found that the expression of Smurf2 was increased with age and in response to regenerative stress during serial transplantation, our findings suggest that Smurf2 plays an important role in regulating HSC self‐renewal and aging.     

Bloodlines of haematopoietic stem cell research in Japan

Haematopoietic stem cells (HSCs) can supply all blood cells throughout the adult life of individuals. Based on this property, HSCs have been used for bone marrow and cord blood transplantation. Among various stem cells, HSCs were recognized earliest and were studied most extensively, providing a model for other stem cells. Knowledge of HSC regulation has rapidly accumulated of late. Contributions of scientists in Japan to progress HSC biology are here briefly overviewed. Focusing on the original work accomplished in Japan in the last two decades, people who have led such activities are introduced and their relationships with one another are sketched.     

Transcriptional regulation of Rex1 (zfp42) in normal prostate epithelial cells and prostate cancer cells

The enormous regenerative capacity of the blood system to sustain functionally mature cells are generated from highly proliferative, short‐lived progenitors, which in turn arise from a rare population of pluripotent and self‐renewing hematopoietic stem cells (HSC). In the bone marrow, these stem cells are kept in a low proliferative, relatively quiescent state in close proximity to stromal cells and osteoblasts, forming specialized niches. The interaction in particular to bone is crucial to prevent exhaustion of HSCs from uncontrolled cell‐cycle entry and to excessive proliferation. In addition, the niche and its components protect stem cells from stress, such as accumulation of reactive oxygen species and DNA damage. One of the key issues is to identify conditions to increase the number of HSCs, either in vivo or during ex vivo growth cultures. This task has been very difficult to resolve and most attempts have been unsuccessful. However, the mechanistic insights to HSC self‐renewal and preservation are gradually increasing and there is now hope that future research will enable scientists and clinicians to modulate the process. In this review, we will focus on the molecular mechanisms of self‐renewal and HSC maintenance in the light of novel findings that HSCs reside at the lowest end of an oxygen gradient. Hypoxia appears to regulate hematopoiesis in the bone marrow by maintaining important HSC functions, such as cell cycle control, survival, metabolism, and protection against oxidative stress. To improve the therapeutic expansion of HSCs we need to learn more about the molecular mechanisms of hypoxia‐mediated regulation. J. Cell. Physiol. 222:17–22, 2010. © 2009 Wiley‐Liss, Inc.     

Leukapheresis cell concentration adjustment required for a successful recovery of HSC after cryopreservation

The recovering of an adequate number of hematopoietic stem cells after cryopreservation is considered pivotal for successful transplantation. Various factors could influence the recovery of HSC following processing and cryopreservation. Therefore, leukapheresis product from thirty patients was cryopreserved in 10% DMSO in cryopreservation bags for their autologous bone marrow transplantation, and 2 ml were cryopreserved in cryovials for post-thaw viability assessment by flow cytometry. The percentage of viable HSCs recovered post-cryopreservation in leukapheresis product was significantly influenced by the concentration of the total nucleated cells cryopreserved per volume. Patients receiving a higher rate of viable HSCs resulted in earlier engraftment of both neutrophils and platelets, so they have been discharged earlier from the hospital. Furthermore, Storage temperature and duration played a role in the recovery of these cells and for the support of the findings, age of the patient at the time of collection did not show any impact on the recovery of this HSC post-cryopreservation. In conclusion, various influencing factors must be taken into consideration during the cryopreservation of HSCs, especially for poor mobilizing patients with a low number of collected hematopoietic stem cells.     

Angiopoietin-Like Protein 3 Promotes Preservation of Stemness during Ex Vivo Expansion of Murine Hematopoietic Stem Cells

Allogeneic hematopoietic stem cell (HSC) transplantations from umbilical cord blood or autologous HSCs for gene therapy purposes are hampered by limited number of stem cells. To test the ability to expand HSCs in vitro prior to transplantation, two growth factor cocktails containing stem cell factor, thrombopoietin, fms-related tyrosine kinase-3 ligand (STF) or stem cell factor, thrombopoietin, insulin-like growth factor-2, fibroblast growth factor-1 (STIF) either with or without the addition of angiopoietin-like protein-3 (Angptl3) were used. Culturing HSCs in STF and STIF media for 7 days expanded long-term repopulating stem cells content in vivo by ∼6-fold and ∼10-fold compared to freshly isolated stem cells. Addition of Angptl3 resulted in increased expansion of these populations by ∼17-fold and ∼32-fold, respectively, and was further supported by enforced expression of Angptl3 in HSCs through lentiviral transduction that also promoted HSC expansion. As expansion of highly purified lineage-negative, Sca-1⁺, c-Kit⁺ HSCs was less efficient than less pure lineage-negative HSCs, Angptl3 may have a direct effect on HCS but also an indirect effect on accessory cells that support HSC expansion. No evidence for leukemia or toxicity was found during long-term follow up of mice transplanted with ex vivo expanded HSCs or manipulated HSC populations that expressed Angptl3. We conclude that the cytokine combinations used in this study to expand HSCs ex vivo enhances the engraftment in vivo. This has important implications for allogeneic umbilical cord-blood derived HSC transplantations and autologous HSC applications including gene therapy.     

Homing and mobilization in the stem cell niche.

All mature blood cells are derived from the haemopoietic stem cell (HSC). In common with all other haemopoietic cells, stem cells are mobile, and it is this property of mobility that has allowed bone marrow transplantation to become a routine clinical option. Successful transplantation requires haemopoietic stem cells to home to the bone marrow, leave the peripheral circulation and become stabilized in regulatory niches in the extravascular space of the bone marrow cavity. This homing and tethering process is reversible - haemopoietic stem cells can be released from their bone marrow tethering through changes in molecular interactions, which are also important in homing following transplantation. The molecular mechanisms regulating this two-way flow of stem cells are beginning to be elucidated, and much recent data has emerged that sheds light on the processes and molecules involved in these complex physiological events. This article reviews current knowledge of the adhesive, homing and proliferative influences acting on HSCs and progenitor cells.     

Hematopoietic capacity of preterm cord blood hematopoietic stem/progenitor cells

Full-term cord blood (TCB) hematopoietic stem/progenitor cells (HSC/HPCs) are used for stem cell transplantation and are well characterized. However, the properties of preterm cord blood (PCB) HSC/HPCs remain unclear. In the present study, we compared HSC/HPCs from TCB and PCB with respect to their expression of surface markers, homing capacity and ability to repopulate HSCs in the NOD/Shi-scid mice bone marrow. The proportion of CD34+CD38− cells was significantly higher in PCB. On the other hand, the engraftment rate of TCB CD34+ cells into NOD/Shi-scid mice was significantly higher than PCB CD34+ cells. The expression of VLA4 was stronger among TCB CD34+ cells than PCB CD34+ cells. Moreover, there was a positive correlation between the proportion of CD34+CXCR4+ cells and gestational age. These data suggest that the homing ability of HSCs increases during gestation, so that TCB may be a better source of HSCs for transplantation than PCB.