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.     

Adoptive transfer of T-cell precursors enhances T-cell reconstitution after allogeneic hematopoietic stem cell transplantation

Immunoincompetence after allogeneic hematopoietic stem cell transplantation (HSCT) affects in particular the T-cell lineage and is associated with an increased risk for infections, graft failure and malignant relapse. To generate large numbers of T-cell precursors for adoptive therapy, we cultured mouse hematopoietic stem cells (HSCs) in vitro on OP9 mouse stromal cells expressing the Notch-1 ligand Delta-like-1 (OP9-DL1). We infused these cells, together with T-cell-depleted mouse bone marrow or purified HSCs, into lethally irradiated allogeneic recipients and determined their effect on T-cell reconstitution after transplantation. Recipients of OP9-DL1-derived T-cell precursors showed increased thymic cellularity and substantially improved donor T-cell chimerism (versus recipients of bone marrow or HSCs only). OP9-DL1-derived T-cell precursors gave rise to host-tolerant CD4+ and CD8+ populations with normal T-cell antigen receptor repertoires, cytokine secretion and proliferative responses to antigen. Administration of OP9-DL1-derived T-cell precursors increased resistance to infection with Listeria monocytogenes and mediated significant graft-versus-tumor (GVT) activity but not graft-versus-host disease (GVHD). We conclude that the adoptive transfer of OP9-DL1-derived T-cell precursors markedly enhances T-cell reconstitution after transplantation, resulting in GVT activity without GVHD.     

Effect of hepatocyte growth factor on long term hematopoiesis of human progenitor cells in transgenic-severe combined immunodeficiency mice

Hepatocyte growth factor (HGF), which was originally isolated as a liver generating factor, enhances hematopoiesis. To study the effect of HGF on hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), we generated severe combined immunodeficiency (SCID) mice producing human (h) HGF and/or stem cell factor (SCF) by transferring the relevant genes to fertilized eggs, and then transplanted hematopoietic progenitors from human cord blood into the transgenic (Tg) SCID mice. Six months after transplantation, a significantly larger number of human cells were found in the Tg SCID mice than in non-Tg controls. Characteristically, the recipient SCID mice producing h HGF (HGF-SCID) had a significantly increased number of h CD41+ cells, whereas the SCF-SCID recipients had more CD11b+ cells. Significantly large numbers of CD34+ progenitors were found in the SCID mice transferred with both h HGF and h SCF genes (HGF/SCF-SCID) when compared with HGF-SCID or SCF-SCID mice. These results imply that HGF supports the differentiation of progenitors in megakaryocyte lineage, whereas SCF supports that in myeloid lineage. The results also imply that HGF acts on HSCs/HPCs as a synergistic proliferative factor combined with SCF. We have demonstrated the advantage of the human cytokine-producing animal in the maintenance of human HSCs.     

Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis

Umbilical cord blood (UCB) transplantation has emerged as a promising therapy, but it is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag's effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, and CD41+ cells in bone marrow compartment without effects on mouse bone marrow cells in the NOD/SCID mice xenotransplant model. Consequently, eltrombopag increased peripheral human platelets and white blood cells. We further examined effects in the STAT and AKT signaling pathways in serum-free cultures. Eltrombopag expanded human CD34+ CD38-, CD34+, and CD41+ cells. Both eltrombopag and recombinant human TPO (rhTPO) induced phosphorylation of STAT5 of CD34+ CD41-, CD34- CD41+, and CD34- CD41- cells. rhTPO preferentially induced pSTAT3, pAKT, and more pSTAT5 in CD34- C41+ cells, while eltrombopag had no effects on pSTAT3. In conclusion, eltrombopag enhanced expansion of HSCs/HPCs of human UCB in vivo and in vitro, and promoted multi-lineage hematopoiesis through the expansion of bone marrow HSCs/HPCs of human UCB in vivo. Eltrombopag differed somewhat from rhTPO in the signal transduction pathways by favoring earlier HSC/HPC populations.     

CD4+ T cells generated de novo from donor hemopoietic stem cells mediate the evolution from acute to chronic graft-versus-host disease

Acute and chronic graft-versus-host disease (GVHD) remain the major complications limiting the efficacy of allogeneic hemopoietic stem cell transplantation. Chronic GVHD can evolve from acute GVHD, or in some cases may overlap with acute GVHD, but how acute GVHD evolves to chronic GVHD is unknown. In this study, in a classical CD8+ T cell-dependent mouse model, we found that pathogenic donor CD4+ T cells developed from engrafted hemopoietic stem cells (HSCs) in C57BL/6SJL(B6/SJL, H-2(b)) mice suffering from acute GVHD after receiving donor CD8+ T cells and HSCs from C3H.SW mice (H-2(b)). These CD4+ T cells were activated, infiltrated into GVHD target tissues, and produced high levels of IFN-gamma. These in vivo-generated CD4+ T cells caused lesions characteristic of chronic GVHD when adoptively transferred into secondary allogeneic recipients and also caused GVHD when administered into autologous C3H.SW recipients. The in vivo generation of pathogenic CD4+ T cells from engrafted donor HSCs was thymopoiesis dependent. Keratinocyte growth factor treatment improved the reconstitution of recipient thymic dendritic cells in CD8+ T cell-repleted allogeneic hemopoietic stem cell transplantation and prevented the development of pathogenic donor CD4+ T cells. These results suggest that de novo-generated donor CD4+ T cells, arising during acute graft-versus-host reactions, are key contributors to the evolution from acute to chronic GVHD. Preventing or limiting thymic damage may directly ameliorate chronic GVHD.     

Progressive divergence of definitive haematopoietic stem cells from the endothelial compartment does not depend on contact with the foetal liver

The yolk sac and the para-aortic splanchnopleura/aorta-genital ridges-mesonephros (P-Sp/AGM) region are the main sites of haematopoietic activity in the mouse embryo at the pre-liver stage of development. By day 11.5 of gestation, the AGM region is capable of autonomous initiation and expansion of definitive haematopoietic stem cells (HSCs). By day 12.5, HSC activity in the AGM region is reduced whilst a second wave of HSCs begins to emerge in the yolk sac. We show here that HSCs emerging in both locations are marked by co-expression of the endothelial-specific marker VE-cadherin and the pan-leukocyte antigen CD45. Phenotypic characterisation using CD31, TIE2, FLK1, Ac-LDL receptors, and CD34 markers demonstrated significant similarities between this VE-cadherin+CD45+ ;double-positive' population and endothelial cells suggesting a common origin for these cells. The double-positive fraction also expressed the stem cell markers Kit, Sca1 and AA4.1. Long-term transplantation experiments demonstrated that the double-positive population, which constituted less than 0.05% of the day 11.5 AGM region and the day 12.5 yolk sac, is highly enriched for HSCs. In vitro assays showed that this population is also enriched for myeloid progenitors. During foetal liver colonization, circulating HSCs remained within the VE-cadherin+ cell fraction, although their phenotypic similarity with endothelial cells became less prominent. Upon liver colonisation the majority of HSCs downregulated VE-cadherin, expression of which was completely lost in the adult bone marrow. Partial loss of VE-cadherin expression in HSCs can be observed extra hepatically in the advanced AGM region by E12.5. Similarly, the CD34+KIT+ population in the placenta, recently identified as a reservoir of HSCs, partly lose VE-cadherin expression by E12.5. By culturing isolated E11.5 AGM region and E12.5 yolk sac we show that the developmental switch from a ;primary' VE-cadherin+CD45+ to a more ;advanced' VE-cadherin-CD45+ phenotype does not require contact of HSCs with the liver and is probably a function of developmental time.     

Cell analysis for hematopoietic stem/progenitor cell transplantation.

Cytometric analysis has become an important aspect in the quality control of cells in all phases of hematopoietic cell transplantation. In the stage of donor conditioning the counting of stem and progenitor cells is important and several reliable single platform tests for CD34+ cells have become available recently. It has been shown, that the count of certain subsets of CD34 may predict best time for harvesting stem cells better than just CD34. In many cases manipulation of the cell sample after collection from the donor is necessary before the cells are adequate for transplantation. Characterization of the resulting cell preparations requires reliable quantitative analysis of a variety of cell types like the enumeration of T-cells at the level of one in ten thousand for some allogeneic transplantations. It is discussed how these clinical requirements will need a refinement of cytometric procedures to achieve adequate clinical decisions.     

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.     

Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding

Disentangling cellular heterogeneity is a challenge in many fields, particularly in the stem cell and cancer biology fields. Here we demonstrate how to combine viral genetic barcoding with high-throughput sequencing to track single cells in a heterogeneous population. We use this technique to track the in vivo differentiation of unitary hematopoietic stem cells (HSCs). The results are consistent with single-cell transplantation studies but require two orders of magnitude fewer mice. In addition to its high throughput, the high sensitivity of the technique allows for a direct examination of the clonality of sparse cell populations such as HSCs. We show how these capabilities offer a clonal perspective of the HSC differentiation process. In particular, our data suggest that HSCs do not equally contribute to blood cells after irradiation-mediated transplantation, and that two distinct HSC differentiation patterns co-exist in the same recipient mouse after irradiation. This technique can be applied to any virus-accessible cell type for both in vitro and in vivo processes.     

An increase in circulating mast cell colony-forming cells in asthma

We compared a potential to generate mast cells among various sources of CD34(+) peripheral blood (PB) cells in the presence of stem cell factor (SCF) with or without thrombopoietin (TPO), using a serum-deprived liquid culture system. From the time course of relative numbers of tryptase-positive and chymase-positive cells in the cultured cells grown by CD34(+) PB cells of nonasthmatic healthy individuals treated with G-CSF, TPO appears to potentiate the SCF-dependent growth of mast cells without influencing the differentiation into mast cell lineage. CD34(+) PB cells from asthmatic patients in a stable condition generated significantly more mast cells under stimulation with SCF alone or SCF+TPO at 6 wk of culture than did steady-state CD34(+) PB cells of normal controls. Single-cell culture studies showed a substantial difference in the number of SCF-responsive or SCF+TPO-responsive mast cell progenitors in CD34(+) PB cells between the two groups. In the presence of TPO, CD34(+) PB cells from asthmatic children could respond to a suboptimal concentration of SCF to a greater extent, compared with the values obtained by those of normal controls. Six-week cultured mast cells of asthmatic subjects had maturation properties (intracellular histamine content and tryptase/chymase enzymatic activities) similar to those derived from mobilized CD34(+) PB cells of nonasthmatic subjects. An increase in a potential of circulating hemopoietic progenitors to differentiate into mast cell lineage may contribute to the recruitment of mast cells toward sites of asthmatic mucosal inflammation.     

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.     

Hematopoietic progenitors express embryonic stem cell and germ layer genes

Cell therapy for tissue regeneration requires cells with high self-renewal potential and with the capacity to differentiate into multiple differentiated cell lineages, like embryonic stem cells (ESCs) and adult somatic cells induced to pluripotency (iPSCs) by genetic manipulation. Here we report that normal adult mammalian bone marrow contains cells, with the cell surface antigen CD34, that naturally express genes characteristic of ESCs and required to generate iPSCs. In addition, these CD34+ cells spontaneously express, without genetic manipulation, genes characteristic of the three embryonic germ layers: ectoderm, mesoderm and endoderm. In addition to the neural lineage genes we previously reported in these CD34+ cells, we found that they express genes of the mesodermal cardiac muscle lineage and of the endodermal pancreatic lineage as well as intestinal lineage genes. Thus, these normal cells in the adult spontaneously exhibit characteristics of embryonic-like stem cells.     

You can count on this: barcoded hematopoietic stem cells

Understanding how individual hematopoietic stem cells contribute to blood formation requires analysis at the single-cell level. Recently in Nature Biotechnology, Lu et al. (2011) tagged HSCs with unique molecular barcodes and used high-throughput sequencing to track their progeny after transplantation.     

Evaluation of intracellular and extracellular trehalose as a cryoprotectant of stem cells obtained from umbilical cord blood

Cord blood is a source of hematopoietic stem cells used in transplantation in which hematopoietic reconstitution is necessary. This transplant modality requires the cryopreservation of hematopoietic stem cells (HSCs). Dimethyl sulfoxide has been used as a cryoprotectant (CPA) in the cryopreservation of HSCs; however, it has been demonstrated that Me₂SO exhibits toxic side effects to the human body. Due to its stability upon freezing, disaccharides such as trehalose have been investigated as a cryoprotectant. This study investigated the hypothesis that a cryopreservation solution containing intracellular and extracellular trehalose improves the recovery of stem cells after cryopreservation. After thawing, the cells were tested for their viability using the 7AAD stain, CD45+/CD34+ cells were assessed using flow cytometry and the MTT viability assay, and the proportion of hematopoietic progenitor cells was measured using the CFU assay. Our results showed the effectiveness of the solution containing intracellular and extracellular trehalose in the cryopreservation of cord blood cells, demonstrating that trehalose may be an optimal cryoprotectant when present both inside and outside of cells.     

Co-culture of human CD34+ cells with mesenchymal stem cells increases the survival of CD34+ cells against the 5-aza-deoxycytidine- or trichostatin A-induced cell death

It has been suggested that epigenetic regulation plays an important role in maintaining the stemness and lineage differentiation of hematopoietic stem cells (HSCs), 5-aza-deoxycytidine (aza-D) and Trichostatin A (TSA) being candidate additives for HSC ex vivo expansion. Although they have potent activity to maintain the stemness, they can also cause serious cell death. This study examined the effects of mesenchymal stem cells (MSCs) on the maintenance of CD34+ cells driven by aza-D and TSA in culture with the combined cytokines of thrombopoietin, flt-3 ligand, stem cell factor, interleukin-3, and interleukin-6. In cultures without MSCs, although aza-D and TSA retained the CD34 frequency 4 to 8 times more than in the cytokines alone, a large portion of cells underwent apoptotic cell death. Consequently, CD34+ cell expansion could not be achieved in any condition without MSCs. In cultures with MSCs, the total cell number was higher in aza-D or TSA than in any conditions in the cultures without MSCs. The CD34 frequency was also similar to the level in the cultures in aza-D or TSA without the MSCs. These results suggest that a co-culture of CD34+ cells with the MSCs might not simply deliver the proliferation signals but also stemness and survival signals, and overlap the action of epigenetic regulators.     

Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment

All blood cell lineages derive from a common hematopoietic stem cell (HSC). The current model implicates that the first lineage commitment step of adult pluripotent HSCs results in a strict separation into common lymphoid and common myeloid precursors. We present evidence for a population of cells which, although sustaining a high proliferative and combined lympho-myeloid differentiation potential, have lost the ability to adopt erythroid and megakaryocyte lineage fates. Cells in the Lin-Sca-1+c-kit+ HSC compartment coexpressing high levels of the tyrosine kinase receptor Flt3 sustain granulocyte, monocyte, and B and T cell potentials but in contrast to Lin-Sca-1+c-kit+Flt3- HSCs fail to produce significant erythroid and megakaryocytic progeny. This distinct lineage restriction site is accompanied by downregulation of genes for regulators of erythroid and megakaryocyte development. In agreement with representing a lymphoid primed progenitor, Lin-Sca-1+c-kit+CD34+Flt3+ cells display upregulated IL-7 receptor gene expression. Based on these observations, we propose a revised road map for adult blood lineage development.     

Purification and functional assay of pluripotent hematopoietic stem cells

Hematolymphopoietic stem cells (HSC) have the capacity for extensive self-renewal and pluripotent myelolymphoid differentiation. Recent studies have emphasized the heterogeneity of human HSC subsets in terms of proliferative and self-renewal capacity. In the NOD-SCID (nonobese diabetic-severe combined immunodeficient) mouse xenograft assay, most CD34+38- stem cell clones proliferate at early times, but then disappear, whereas only few clones persist: possibly, the latter ones consist of long-term engrafting CD34+38- HSC expressing the KDR receptor (i.e. the vascular endothelial growth factor receptor II). In this regard, isolation of the small KDR+ subset from the CD34+ hematopoietic progenitors (and possibly from the CD34-lin- population) may provide a novel and effective approach for the purification of long-term proliferating HSC. More importantly, KDR+ HSC isolation will pave the way to cellular/molecular characterization and improved functional manipulation of HSC/HSC subsets, as well as to innovative approaches for HSC clinical utilization, specifically transplantation, transfusion medicine and gene therapy.     

Enhancement of proliferation of human umbilical cord blood–derived CD34+ hematopoietic stem cells by a combination of hyper-interleukin-6 and small molecules

Umbilical cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cells (HSCs) for transplantation to treat various hematological disorders. The major limitation to the use of UCB-derived HSCs (UCB–HSCs) in transplantation, however, is the low numbers of HSCs in a unit of cord blood. To overcome this limitation, various cytokines or small molecules have been used to expand UCB-HSCs ex vivo. In this study, we investigated a synergistic effect of the combination of HIL-6, SR1, and UM171 on UCB-HSC culture and found that this combination resulted in the highest number of CD34⁺ cells. These results suggest that the combination of SR1, UM171 and HIL-6 exerts a synergistic effect in the proliferation of HSCs from UCB and thus, SR1, UM171 and HIL-6 is the most suitable combination for obtaining HSCs from UCB for clinical transplantation.