Ex vivo expanded umbilical cord blood T cells maintain naive phenotype and TCR diversity

BACKGROUND: Umbilical cord blood (CB) is a promising source of hematopoietic stem cells for allogeneic transplantation. However, delayed engraftment and impaired immune reconstitution remain major limitations. Enrichment of donor grafts with CB T cells expanded ex vivo might facilitate improved T-cell immune reconstitution post-transplant. We hypothesized that CB T cells could be expanded using paramagnetic microbeads covalently linked to anti-CD3 and anti-CD28 Ab. METHODS: CB units were divided into three fractions: (1) cells cultured without beads, (2) cells cultured with beads and (3) cells cultured with beads following CD3+ magnetic enrichment. All fractions were cultured for 14 days in the presence of IL-2 (200 IU/mL). RESULTS: A mean 100-fold expansion (range 49-154) of total nucleated cells was observed in the CD3+ magnetically enriched fraction. Following expansion, CB T cells retained a naive and/or central memory phenotype and contained a polyclonal TCR diversity demonstrated by spectratyping. DISCUSSION: Our data provide evidence that naive and diverse CB T cells may be expanded ex vivo and warrant additional studies in the setting of human CB transplantation.     

Co-culture of cord blood CD34(+) cells with human BM mesenchymal stromal cells enhances short-term engraftment of cord blood cells in NOD/SCID mice

BACKGROUND: The major challenge for cord blood transplantation (CBT) is higher rates of delayed and failed engraftment. In an attempt to broaden the application of CBT to more candidates, ex vivo expansion of hematopoietic stem/progenitor cells in CB is a major area of investigation. The purpose of this study was to employ human BM mesenchymal stromal cells (hBM-MSC) as the feeding-layer to expand CB cells ex vivo. METHODS: In this study, hBM-MSC were isolated and characterized by morphologic, mmunophenotypic and RT-PCR analysis. The hBM-MSC at passage 3 were employed as the feeding-layer to expand CB CD34(+) cells in vivo in the presence of thrombopoietin, flt3/flk2 ligand, stem cell factor and G-CSF. The repopulating capacity of the ex vivo-expanded CB cells was also evaluated in a NOD/SCID mice transplant experiment. RESULTS: After 1 or 2 weeks of in vitro expansion, hBM-MSC supported more increasing folds of CB in total nucleated cells, CD34(+) cells and colony-forming units (CFU) compared with CB without hBM-MSC. Furthermore, although NOD/SCID mice transplanted with CB cells expanded only in the presence of cytokines showed a higher percentage of human cell engraftment in BM than those with unexpanded CB CD34(+) cells, expanded CB cells co-cultured with hBM-MSC were revealed to enhance short-term engraftment further in recipient mice. DISCUSSION: Our study suggests that hBM-MSC enhance in vitro expansion of CB CD34(+) cells and short-term engraftment of expanded CB cells in NOD/SCID mice, which may be valuable in a clinical setting.     

Ex vivo expansion of cord blood mononuclear cells on mesenchymal stem cells

BACKGROUND: Cord blood (CB) cells are being used increasingly as a source of hematopoietic cells to support high dose chemotherapy. However, CB units contain low numbers of cells, including CD34+ cells, and thus their use is associated with significant delays in engraftment of neutrophils and platelets. Exvivo expansion of CB has been proposed to increase the numbers of cells available. We and others have reported the requirement of CD34 selection for optimal expansion of CB products'; however, the selection of frozen CB products in clinical trials results in significant loss of CD34+ cells, with a median recovery of 50, but less than 40% recovery in more than one-third of products. In the present studies we evaluated the potential of mesenchymal stem cells (MSC) to support ex vivo expansion of unselected CB products. METHODS: Mononuclear cells (MNC) from CB products were isolated and cultured on preformed MSC layers in T150 flasks containing 50 mL Stemline II media plus hematopoietic growth factors. Various culture conditions were compared for optimal expansion of the CB MNC. RESULTS: Ex vivo expansion of CB MNC on MSC resulted in 10- to 20-fold expansion of total nucleated cells, seven- to 18-fold expansion of committed progenitor cells, two- to five-fold expansion of primitive progenitor cells and 16- to 37-fold expansion of CD34+ cells. DISCUSSION: These studies demonstrated significant expansion of CB products without CD34 cell selection using culture conditions that are clinically applicable. Our current focus is to initiate clinical trials to evaluate the in vivo potential of CB cells expanded with these conditions.     

Ex vivo expansion and clonality of CD34+ selected cells from bone marrow and cord blood in a serum-free media

Although umbilical cord blood is increasingly being used in allogeneic marrow transplantation, delayed platelet engraftment is often a concern for cord blood transplant recipients. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from a bone marrow source, and cord blood, in a serum-free Media. The CD34+ cells, selected from bone marrow (BM) and umbilical cord blood (CB), were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at days 0, 4, 7, and 14 under the combination of growth factors, with cell counts. The cytokines included the recombinant human megakaryocyte growth and development (100 ng/ml), interleukin-3 (10 ng/ml), stem cell factor (100 ng/ml), flt-3 ligand (50 ng/ml) and interleukin-11 (200 ng/ml). The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the BM at day 7 (3.0 fold increase than BM), day 14 (2.4 fold), and day 17 (2.6 fold). The colony count of the BFU-E/CFU-E per CD34+ cell at day 0 was 0.14 +/- 0.023 in the CB, which was significantly higher than 0.071 +/- 0.015 in the BM. The CB-selected CD34+ cells produced more BFU-E colonies than the BM on culture days 4, 7, and 14. The BFU-E colonies from the CB cells increased markedly on culture days 4 and 7, with a 4-fold increase at day 14. The colony count of the CFU-Mk per CD34+ cell at day 0 was 0.047 +/- 0.011 in the CB-selected CD34+ cells cultures, which was higher than the 0.026 +/- 0.014 in the BM. The CB-selected CD34+ cells produced more CFU-Mk colonies than the BM on culture days 4, 7 and 14. In conclusion, the ex vivo expansion of the CB cells may be very promising in producing total cellular expansion, CFU-Mk and BFU-E compared with BM, especially at day 7. The ex vivo expansion of the CB may have rationale in making an ex vivo culture for 7 to 14 d.     

A novel direct competitive repopulation assay for human hematopoietic stem cells using NOD/SCID mice

BACKGROUND: The major problem in cord blood (CB) transplantation for adult patients is shortage of stem cell number. To overcome this disadvantage, several studies on ex vivo expansion have been performed. However, such efforts are always troubled by the lack of a reliable and simple assay system for stem cells. Our aim was to establish an in vivo assay system to compare the directly repopulating ability of two populations of human hematopoietic stem cells using a xenogeneic transplant system. METHODS: Thirty CB samples from infants of each sex were pooled and enriched for CD34(+) progenitor cells. Enriched CD34(+) cells were transplanted into irradiated NOD/SCID mice at different male to female ratios, and human hematopoietic cells recovered 7 weeks after transplantation were analyzed by a quantitative DNA sex test using competitive PCR for the amelogenin gene. Using this assay system, ex vivo cultured and non-cultured CB cells were compared for repopulating ability. RESULTS: The sex ratio of human CB cells transplanted was found to be maintained for 7 weeks in matured and progenitor cells. The competitive repopulation assay of cultured and non-cultured CB cells showed a marked defect in the repopulating ability of cultured cells, although the LTCIC count was maintained during cultivation. DISCUSSION: Our assay system is a simple and reliable quantitative method that permits direct comparison of two stem cell compartments. The assay system will be useful for the assessment of the functional abilities of various human hematopoietic stem cells.     

Pre-clinical development of cord blood-derived progenitor cell graft expanded ex vivo with cytokines and the polyamine copper chelator tetraethylenepentamine

BACKGROUND: We have previously demonstrated that the copper chelator tetraethylenepentamine (TEPA) enables preferential expansion of early hematopoietic progenitor cells (CD34+CD38-, CD34+CD38-Lin-) in human umbilical cord blood (CB)-derived CD34+ cell cultures. This study extends our previous findings that copper chelation can modulate the balance between self-renewal and differentiation of hematopoietic progenitor cells. METHODS: In the present study we established a clinically applicative protocol for large-scale ex vivo expansion of CB-derived progenitors. Briefly, CD133+ cells, purified from CB using Miltenyi Biotec's (Bergisch Gladbach, Germany) CliniMACS separation device and the anti-CD133 reagent, were cultured for 3 weeks in a clinical-grade closed culture bag system, using the chelator-based technology in combination with early-acting cytokines (SCF, thrombopoietin, IL-6 and FLT-3 ligand). This protocol was evaluated using frozen units derived from accredited cord blood banks. RESULTS: Following 3 weeks of expansion under large-scale culture conditions that were suitable for clinical manufacturing, the median output value of CD34+ cells increase by 89-fold, CD34+CD38- increase by 30-fold and CFU cells (CFUc) by 172-fold over the input value. Transplantation into sublethally irradiated non-obese diabetic (NOD/SCID) mice indicated that the engraftment potential of the ex vivo expanded CD133+ cells was significantly superior to that of unexpanded cells: 60+/-5.5% vs. 21+/-3.5% CD45+ cells, P=0.001, and 11+/-1.8% vs. 4+/-0.68% CD45+CD34+ cells, P=0.012, n=32, respectively. DISCUSSION: Based on these large-scale experiments, the chelator-based ex vivo expansion technology is currently being tested in a phase 1 clinical trial in patients undergoing CB transplantation for hematological malignancies.     

Hematopoietic reconstitution of CD34+ cells derived from short-term cultured cord blood mononuclear cells

Ex vivo expansion of hematopoietic stem cells (HSCs) is very important for clinical applications of cord blood (CB). With the aim to find proper culture duration for ex vivo expansion, mononuclear cells (MNC) was applied as starting culture cells to expand HSCs and the repopulating potential of seven-day and fourteen-day cultured CD34⁺ cells were compared. The average expansion of total cells and CD34⁺ cells cultured for 7 days were higher than those cultured for 14 days. The results of phenotypic analysis of fresh and cultured cells showed that the percentage of CD3⁺ cells declined and the percentage of CD33⁺ cells increased during culture. The engraftment levels of fourteen-day cultured CD34⁺ cells were higher than those of fresh and seven-day cultured CD34⁺ cells. Fourteen-day cultured CD34⁺ cells also showed better multilineage reconstitution ability than fresh and seven-day cultured CD34⁺ cells. The results of the present study demonstrated that prolonged culture could preserve the hematopoietic reconstitution ability of ex vivo cultured CB cells and improve the engraftment level in NOD/SCID mice.     

Expansion of Cord Blood CD34+ Cells in Presence of zVADfmk and zLLYfmk Improved Their In Vitro Functionality and In Vivo Engraftment in NOD/SCID Mouse

Background

Cord blood (CB) is a promising source for hematopoietic stem cell transplantations. The limitation of cell dose associated with this source has prompted the ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs). However, the expansion procedure is known to exhaust the stem cell pool causing cellular defects that promote apoptosis and disrupt homing to the bone marrow. The role of apoptotic machinery in the regulation of stem cell compartment has been speculated in mouse hematopoietic and embryonic systems. We have consistently observed an increase in apoptosis in the cord blood derived CD34⁺ cells cultured with cytokines compared to their freshly isolated counterpart. The present study was undertaken to assess whether pharmacological inhibition of apoptosis could improve the outcome of expansion.

Methodology/Principal Findings

CB CD34⁺ cells were expanded with cytokines in the presence or absence of cell permeable inhibitors of caspases and calpains; zVADfmk and zLLYfmk respectively. A novel role of apoptotic protease inhibitors was observed in increasing the CD34⁺ cell content of the graft during ex vivo expansion. This was further reflected in improved in vitro functional aspects of the HSPCs; a higher clonogenicity and long term culture initiating potential. These cells sustained superior long term engraftment and an efficient regeneration of major lympho-myeloid lineages in the bone marrow of NOD/SCID mouse compared to the cells expanded with growth factors alone.

Conclusion/Significance

Our data show that, use of either zVADfmk or zLLYfmk in the culture medium improves expansion of CD34⁺ cells. The strategy protects stem cell pool and committed progenitors, and improves their in vitro functionality and in vivo engraftment. This observation may complement the existing protocols used in the manipulation of hematopoietic cells for therapeutic purposes. These findings may have an impact in the CB transplant procedures involving a combined infusion of unmanipulated and expanded grafts.     

Enhancing bone marrow regeneration by SALL4 protein

Hematopoietic stem cells (HSCs) are widely used in transplantation therapy to treat a variety of blood diseases. The success of hematopoietic recovery is of high importance and closely related to the patient’s morbidity and mortality after Hematopoietic stem cell transplantation (HSCT). We have previously shown that SALL4 is a potent stimulator for the expansion of human hematopoietic stem/progenitor cells in vitro. In these studies, we demonstrated that systemic administration with TAT-SALL4B resulted in expediting auto-reconstitution and inducing a 30-fold expansion of endogenous HSCs/HPCs in mice exposed to a high dose of irradiation. Most importantly, TAT-SALL4B treatment markedly prevented death in mice receiving lethal irradiation. Our studies also showed that TAT-SALL4B treatment was able to enhance both the short-term and long-term engraftment of human cord blood (CB) cells in NOD/SCID mice and the mechanism was likely related to the in vivo expansion of donor cells in a recipient. This robust expansion was required for the association of SALL4B with DNA methyltransferase complex, an epigenetic regulator critical in maintaining HSC pools and in normal lineage progression. Our results may provide a useful strategy to enhance hematopoietic recovery and reconstitution in cord blood transplantation with a recombinant TAT-SALL4B fusion protein.     

Fucosylation with fucosyltransferase VI or fucosyltransferase VII improves cord blood engraftment

Background aimsAdvantages associated with the use of cord blood (CB) transplantation include the availability of cryopreserved units, ethnic diversity and lower incidence of graft-versus-host disease compared with bone marrow or mobilized peripheral blood. However, poor engraftment remains a major obstacle. We and others have found that ex vivo fucosylation can enhance engraftment in murine models, and now ex vivo treatment of CB with fucosyltransferase (FT) VI before transplantation is under clinical evaluation (NCT01471067). However, FTVII appears to be more relevant to hematopoietic cells and may alter acceptor substrate diversity. The present study compared the ability of FTVI and FTVII to improve the rapidity, magnitude, multi-lineage and multi-tissue engraftment of human CB hematopoietic stem and progenitor cells (HSPCs) in vivo.MethodsCD34-selected CB HSPCs were treated with recombinant FTVI, FTVII or mock control and then injected into immunodeficient mice and monitored for multi-lineage and multi-tissue engraftment.ResultsBoth FTVI and FTVII fucosylated CB CD34⁺ cells in vitro, and both led to enhanced rates and magnitudes of engraftment compared with untreated CB CD34⁺ cells in vivo. Engraftment after treatment with either FT was robust at multiple time points and in multiple tissues with similar multi-lineage potential. In contrast, only FTVII was able to fucosylate T and B lymphocytes.ConclusionsAlthough FTVI and FTVII were found to be similarly able to fucosylate and enhance the engraftment of CB CD34⁺ cells, differences in their ability to fucosylate lymphocytes may modulate graft-versus-tumor or graft-versus-host effects and may allow further optimization of CB transplantation.     

Biology of cord blood cells and future prospects for enhanced clinical benefit

Cord blood (CB) has served as a clinically beneficial source of hematopoietic stem (HSC) and progenitor (HPC) cells for transplantation and correction of a large number of malignant and non-malignant disorders. The capacity of CB to perform these functions is intimately related to the quality and quantity of HSC and HPC present in CB. This review covers the biology of HSC and HPC, efforts to expand these cells ex vivo for enhanced clinical utility that has thus far not been very successful, and recent studies on attempts to enhance the homing and engrafting capability of HSC as an alternative means for more effective use of the limited numbers of CB cells collected. This review also highlights the presence in CB of mesenchymal stem cells, unrestricted somatic stem cells, endothelial progenitor cells and immune cells. The presence and biology of these non-HSC/HPC may open up future possibilities for additional clinical benefit of CB, a product considered mainly for discard before its clinical transplantation potential was realized in the late 1980s.     

人脐带血来源造血干细胞体外扩增的研究进展

造血干细胞（HSCs）是血液系统中的一类成体干细胞群,具有自我更新和多谱系分化两个基本特征。造血干细胞移植（HSCT）可以治疗退行性疾病和多种血液系统疾病。脐带血来源造血干细胞（CB HSCs）是降低HLA配型要求的突破点,但单份脐带血中HSCs数量不能满足使用要求,为了获得足够数量的CB HSCs,体外扩增是一种可行的方法。近几年,学者们探索了多种体外扩增方法,包括优化细胞生长因子混合物、与基质细胞共培养及加入小分子化合物（SMCs）激动剂等。目前应用细胞因子联合小分子的扩增方法在多个临床试验中获得成功。本文对目前体外扩增CB HSCs的研究进展做一综述。     

Overcoming the barriers to umbilical cord blood transplantation

Umbilical cord blood (UCB) transplantation (UCBT) has seen a marked increase in utilization in recent years, especially in the pediatric population; however, graft failure, delayed engraftment and profound delay in immune reconstitution leads to significant morbidity and mortality in adults. The lack of cells available for post-transplant therapies, such as donor lymphocyte infusions, has also been considered a disadvantage. To overcome the cell–dose barrier, the combination of two UCB units is becoming commonplace in adolescent and adult populations, and is currently being studied in pediatrics as well. In some studies, the use of two UCB units appears to have a positive impact on outcomes; however, engraftment is still suboptimal. A possible additional way to improve outcome and extend applicability of UCBT is via ex vivo expansion. Studies to develop optimal expansion conditions are still in the exploratory phase; however, recent studies suggest expanded UCB is safe and can improve outcomes. The ability to transplant across HLA disparities, rapid procurement time and decreased graft-versus-host disease (GvHD) seen with UCBT makes it a promising stem cell source and, while barriers exist, consistent progress is being made to overcome them.     

Increased megakaryopoiesis in cultures of CD34-enriched cord blood cells maintained at 39 degrees C

Based on previous evidence suggesting positive effects of fever on in vivo hematopoiesis, we tested the effect of hyperthermia on megakaryopoiesis (MK) in ex vivo cultures of CD34-enriched cord blood (CB) cells. The cells were cultured at 37 degrees C or 39 degrees C for 14 days in cytokine conditions optimized for megakaryocyte development and analyzed periodically. Compared to 37 degrees C, the cultures maintained at 39 degrees C produced significantly more (up to 10-fold) total cells, myeloid and MK progenitors, and total MKs, and showed accelerated and enhanced MK maturation with increased yields of proplatelets and platelets. This observation could facilitate clinical applications requiring ex vivo expansion of hematopoietic cells.     

Unrelated and related cord blood banking and hematopoietic graft engineering

The first successful transplantation of umbilical-cord blood (CB) was performed in 1988 to treat a boy with Fanconi's anemia, using CB from his HLA full-matched sister. A few years later, CB transplantation (CBT) was also performed in an adult recipient, however major obstacles still prevent a wider application of CBT in this age group. The principle limiting-factor is the low numbers of nucleated (NC) and CD34+ cells available for transplantation compared to a typical bone marrow (BM)/peripheral blood (PB) allograft, resulting in a lower engraftment success as well as delayed hematopoietic recovery with its characteristic complications, including infections and transplant related mortality (TRM). Other problems include uncertainty regarding potency and efficacy of graft versus leukemia (GvL)/tumor effects in this kind of transplant, considering the reduced graft versus host disease (GvHD) manifestations and immunologic prematurity. These subjects are reviewed with orientation to technical methods directed to improve CB grafts and graft engineering.     

Low rate of infusional toxicity after expanded cord blood transplantation

Background aimsUmbilical cord blood (CB) is used with increasing frequency to restore hematopoiesis in patients with bone marrow transplant who lack a suitable human leukocyte antigen–matched donor. CB transplantation is limited by low cell doses and delays in neutrophil and platelet engraftment. CB progenitors expanded ex vivo before transplantation provide more rapid hematopoietic and immune reconstitution as well as less engraftment failure compared with unmanipulated CB. However, the safety of infusing double and ex vivo–expanded CB has not been systematically examined.MethodsWe reviewed the immediate adverse events (AE) associated with the infusion of CB occurring within 24 hours in 137 patients enrolled in clinical CB transplant trials at the MD Anderson Cancer Center from February 2004 to May 2010. All patients received an unmanipulated CB unit followed by infusion of a second unmanipulated CB unit or a second CB unit expanded ex vivo with the use of cytokines in a liquid culture system or in mesenchymal stromal cell co-cultures.ResultsA total of three grade 2 and two grade 3 infusion reactions occurred within 24 hours of CB transplantation. This resulted in an AE rate of 3.7%. The majority of AEs manifested as signs of hypertension. No association with patient age, sex, disease status, premedication, ABO compatibility or total infusion volume was observed. In summary, the incidence of infusion-related toxicities in patients who receive unmanipulated and ex vivo–expanded double CB transplantation is low.ConclusionsWe conclude that the infusion of unmanipulated followed by expanded CB products is a safe procedure associated with a low probability of inducing severe reactions.     

Expansion of megakaryocyte progenitors from cryopreserved leukocyte concentrates of human placental and umbilical cord blood in short-term liquid culture

Long-term severe thrombocytopenia following human placental and umbilical cord blood (CB) transplantation is a significant clinical problem. We studied the ex vivo expansion of megakaryocytic progenitor cells (CFU-Meg) from cryopreserved/thawed leukocyte concentrates (LC) of CB prepared by the Tokyo Cord Blood Bank protocol. The LC cells were cultured in serum-free culture medium supplemented with a combination of early-acting cytokines including thrombopoietin (TPO), flt3-ligand (FL), and stem cell factor (SCF). Combination of TPO plus FL, TPO plus SCF, and all of these cytokines together resulted in 8.9-, 7.7-, and 8.4-fold increases in CFU-Meg, respectively, by Day 5 of culture. Our results showed that this simple expansion strategy has the potential for expanding CFU-Meg from cryopreserved/thawed LC cells from CB.     

Hematopoietic stem cell expansion: challenges and opportunities

Attempts to improve hematopoietic reconstitution and engraftment potential of ex vivo-expanded hematopoietic stem and progenitor cells (HSPCs) have been largely unsuccessful due to the inability to generate sufficient stem cell numbers and to excessive differentiation of the starting cell population. Although hematopoietic stem cells (HSCs) will rapidly expand after in vivo transplantation, experience from in vitro studies indicates that control of HSPC self-renewal and differentiation in culture remains difficult. Protocols that are based on hematopoietic cytokines have failed to support reliable amplification of immature stem cells in culture, suggesting that additional factors are required. In recent years, several novel factors, including developmental factors and chemical compounds, have been reported to affect HSC self-renewal and improve ex vivo stem cell expansion protocols. Here, we highlight early expansion attempts and review recent development in the extrinsic control of HSPC fate in vitro.     

Improved hematopoietic stem cell engraftment following ex vivo expansion of murine marrow cells with SCF and Flt3L

BACKGROUND: In vitro incubation of murine BM cells with IL-3, IL-6, IL-11 and SCF induces expansion of HPC but fails to preserve 'engraftability' in comparison with normal untreated marrow cells. We studied how culturing marrow cells for 48 and 72 h with a combination of the cytokines SCF and Flt3L influences cell expansion and engraftability. METHODS: Competitive repopulation of lethally irradiated C57BL/6 mice was used to examine engraftability of ex vivo cytokine-expanded Ptprc chimeric BM. A methylcellulose in vitro assay was used to determine the expansion of substitute progenitors. RESULTS: Both cytokine combinations successfully expanded progenitor populations when assayed in methylcellulose culture in vitro. After 72 h, the colony numbers of the expansion cultures increased 61% with IL-3, IL-6, IL-11 and SCF stimulation and 96% with SCF and Flt3L stimulation. Engraftment of competitively transplanted cells, cultured with IL-3, IL-6, IL-11 and SCF, consistently dropped to levels below 16%. However, 48 h culture with SCF and Flt3L resulted in 53.5+/-1.6% engraftment at 17 days and 64+/-3.7% engraftment at 19 weeks post-transplantation. Extending the cytokine exposure to 72 h resulted in 70+/-4.4% short-term engraftment at 17 days, and 64+/-2.4% engraftment at 19 weeks post-transplantation. DISCUSSION: The data demonstrate the ability of SCF and Flt3L cytokine-stimulated BM cells to maintain short- and long-term engraftability. We conclude that these cytokines play a crucial role in maintaining engraftment of hematopoietic progenitors.