Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice

OBJECTIVE: For approximately 5% of autologous transplant recipients and a higher proportion of allogeneic transplant recipients, low level and delayed platelet engraftment is an ongoing problem. Mesenchymal stem cells (MSC), which can be derived from bone marrow as well as other organs, are capable of differentiation into multiple cell types and also support hematopoiesis in vitro. Because cotransplantation of marrow-derived stromal cells has been shown to enhance engraftment of human hematopoietic stem cells, we hypothesized that cotransplantation of MSC could enhance platelet and myeloid cell development. MATERIALS AND METHODS: We tested this hypothesis by transplantation of CD34-selected mobilized human peripheral blood stem cells (PBSC) into sublethally irradiated NOD/SCID mice with or without culture-expanded human MSC and evaluated human myeloid, lymphoid, and megakaryocytic engraftment with flow cytometry and in vitro cultures. RESULTS: We find that MSC cotransplantation enhances human cell engraftment when a limiting dose (<1 x 10(6)) of CD34 cells is administered. This enhancement is characterized by a shift in the differentiation of human cells from predominantly B lymphocytes to predominantly CD13(+), CD14(+), and CD33(+) myeloid cells with a corresponding increase in myeloid CFU in the marrow. Megakaryocytopoiesis is enhanced by MSC cotransplantation as assessed by an increase in both marrow CFU-MK and circulating human platelets. In contrast, MSC do not affect the percentage of human bone marrow cells that expresses CD34(+). CONCLUSIONS: Cotransplantation of human mesenchymal stem cells with CD34(+)-selected hematopoietic stem cells enhances myelopoiesis and megakaryocytopoiesis.     

Mesenchymal stem cells promote engraftment of human umbilical cord blood-derived CD34(+) cells in NOD/SCID mice

OBJECTIVE: Mesenchymal stem cells (MSC) have been implicated as playing an important role in hematopoietic stem cell engraftment. We identified and characterized a new population of MSC derived from human fetal lung. In cotransplantation experiments, we examined the homing of MSC as well as the effect on engraftment of human umbilical cord blood (UCB)-derived CD34(+) cells in NOD/SCID mice. MATERIALS AND METHODS: Culture-expanded fetal lung-derived CD34(+) cells were characterized by immune phenotyping and cultured under conditions promoting differentiation to osteoblasts or adipocytes. Irradiated (3.5 Gy) NOD/SCID mice (n = 51) were transplanted intravenously with 0.03 to 1.0 x 10(6) UCB CD34(+) cells in the presence or absence of 1 x 10(6) culture-expanded fetal lung-derived MSC, irradiated CD34(-) cells, B cells, or with cultured MSC only. RESULTS: Culture-expanded fetal lung CD34(+) cells were identified as MSC based on phenotype (CD105(+), SH3(+), SH4(+), CD160(+)) and their multilineage potential. Cotransplantation of low doses of UCB CD34(+) cells and MSC resulted in a three-fold to four-fold increase in bone marrow engraftment after 6 weeks, whereas no such effect was observed after cotransplantation of irradiated CD34(-) or B cells. Homing experiments indicated the presence of MSC in the lung, but not in the bone marrow, of NOD/SCID mice. CONCLUSIONS: We identified a population of MSC derived from human fetal lung. Upon cotransplantation, MSC, but not irradiated CD34(-) or B cells, promote engraftment of UCB CD34(+) cells in bone marrow, spleen, and blood by mechanisms that may not require homing of MSC to the bone marrow.     

Reversible expression of CD34 by adult human bone marrow long-term engrafting hematopoietic stem cells

OBJECTIVE: We previously reported that CD34(-) population of bone marrow (BM) cells from adult humans contains cells capable of engraftment and multilineage differentiation. We also reported on the reversibility of CD34 expression by murine hematopoietic stem cells. Based on long-term observations in primary, secondary, and tertiary sheep recipients, we now present definitive evidence for the long-term engrafting capability of human BM CD34(-) cells, and the reversibility of CD34 expression by human BM hematopoietic stem cells (HSC) in vivo. MATERIALS AND METHODS: We used serial transplantations into primary, secondary, and tertiary preimmune fetal sheep recipients to evaluate and compare the long-term engraftment and differentiation of adult human bone marrow-derived CD34(-) and CD34(+) cells in vivo. RESULTS: In primary hosts CD34(-) or CD34(+) cells produced multilineage human cell activity that persisted for 31 months. To confirm the long-term engrafting characteristics of CD34(-) cells and determine whether CD34 expression on human HSC is reversible, we transplanted human CD34(-) and CD34(+) cells obtained from primary hosts into secondary sheep recipients. Multilineage engraftment occurred in all secondary hosts, and in tertiary hosts transplanted with CD34(-) or CD34(+) cells obtained from BM of secondary recipients. CONCLUSION: These results demonstrate that human BM CD34(-) cells are capable of long-term multilineage engraftment in vivo. The finding that both CD34(-) and CD34(+) cells from primary/secondary groups engraft secondary/tertiary hosts indicates that CD34 expression on human HSC is reversible, a process that does not impair HSC function in vivo.     

Reversible expression of CD34 by murine hematopoietic stem cells.

We used a mouse transplantation model to address the recent controversy about CD34 expression by hematopoietic stem cells. Cells from Ly-5.1 C57BL/6 mice were used as donor cells and Ly-5.2 mice were the recipients. The test cells were transplanted together with compromised marrow cells of Ly-5.2 mice. First, we confirmed that the majority of the stem cells with long-term engraftment capabilities of normal adult mice are CD34(-). We then observed that, after the injection of 150 mg/kg 5-fluorouracil (5-FU), stem cells may be found in both CD34(-) and CD34(+) cell populations. These results indicated that activated stem cells express CD34. We tested this hypothesis also by using in vitro expansion with interleukin-11 and steel factor of lineage(-) c-kit(+) Sca-1(+) CD34(-) bone marrow cells of normal mice. When the cells expanded for 1 week were separated into CD34(-) and CD34(+) cell populations and tested for their engraftment capabilities, only CD34(+) cells were capable of 2 to 5 months of engraftment. Finally, we tested reversion of CD34(+) stem cells to CD34(-) state. We transplanted Ly-5.1 CD34(+) post-5-FU marrow cells into Ly-5.2 primary recipients and, after the marrow achieved steady state, tested the Ly-5.1 cells of the primary recipients for their engraftment capabilities in Ly-5.2 secondary recipients. The majority of the Ly-5.1 stem cells with long-term engraftment capability were in the CD34(-) cell fraction, indicating the reversion of CD34(+) to CD34(-) stem cells. These observations clearly demonstrated that CD34 expression reflects the activation state of hematopoietic stem cells and that this is reversible.     

Loss of marrow reserve from dose-intensified chemotherapy results in impaired hematopoietic reconstitution after autologous transplantation: CD34(+), CD34(+)38(-), and week-6 CAFC assays predict poor engraftment

Autologous hematopoietic stem cell transplantation (HSCT) is an increasingly successful modality for treating a variety of malignant disorders in the clinic. Experimental and clinical data suggest that prior exposure to cytotoxic agents that damage primitive stem cells results in impaired hematopoiesis after autologous HSCT. To further investigate the ability to predict for impaired hematopoiesis, we measured different stem/progenitor cell populations transplanted and time to engraftment. Patients with previously untreated, advanced-stage follicular lymphoma were treated in sequential prospective protocols with 6-8 cycles of standard-dose (SD) cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or four cycles of a higher-dose (HD) CHOP and granulocyte colony-stimulating factor, to induce remission prior to high-dose cyclophosphamide, total body irradiation, and autologous bone marrow transplantation (ABMT). Cryopreserved marrow samples obtained prior to ABMT were assayed for CD34(+), CD34(+)38(-), and cobblestone area-forming cell (CAFC) frequencies.Despite receiving similar numbers of nucleated cells at ABMT, HD-CHOP patients took significantly longer to attain platelet engraftment than the SD-CHOP patients. Marrow from the HD-CHOP patients contained significantly lower CD34(+), CD34(+)38(-), and week 6-8 CAFC frequencies than marrow from SD-CHOP-treated patients. Time to platelet engraftment was plotted against progenitor/stem cell numbers transplanted for each patient and threshold values were developed for all three stem/progenitor cell populations. These values were 0.5 x 10(6) CD34(+) cells/kg, 0.14 x 10(6) CD34(+)38(-) cells/kg, and 9500 week-6 CAFC/kg transplanted. Approximately 50% of patients received marrow progenitor/stem cell numbers above the threshold values and all engrafted without delay. However, transplantation of stem/progenitor cell numbers below threshold values did not uniformly predict for delayed platelet engraftment.These data provide further evidence for the association of low marrow reserve at ABMT, low numbers of stem/progenitor cells transplanted, and delayed hematopoietic recovery. However, there remains a group of patients who have rapid platelet engraftment after ABMT despite low numbers of progenitor/stem cells transplanted. These data suggest the presence of a crucial stem cell population not represented by the stem/progenitor cell populations studied in these experiments.     

CD45 regulates homing and engraftment of immature normal and leukemic human cells in transplanted immunodeficient mice

Bone marrow homing and engraftment by clinically transplanted hematopoietic stem and progenitor cells is a complex process that is not fully understood. We report that the pan-leukocyte CD45 phosphatase plays an essential role in trafficking and repopulation of the bone marrow by immature human CD34(+) cells and leukemic cells in transplanted nonobese diabetic severe combined immunodeficient mice. Inhibiting CD45 function by blocking antibodies or a CD45 inhibitor impaired the motility of both normal and leukemic human cells. Blocking CD45 inhibited homing and repopulation by immature human CD34(+) cells as well as homing of primary patient leukemic cells. In addition, CD45 inhibition negatively affected development of hematopoietic progenitors in?vitro and their recovery in transplanted recipients in?vivo, revealing the central role of CD45 in the regulation of hematopoiesis. Moreover, CD45 blockage induced a hyperadhesive phenotype in immature human progenitor cells as well as in murine leukocytes, leading to their defective adhesion interactions with endothelial cells. This phenotype was further manifested by the ability of CD45 blockage to prevent breakdown of adhesion interactions in the BM, which inhibited murine progenitor mobilization. The substantial effects of a direct CD45 inhibition point at its essential roles in cell trafficking, including murine progenitor cell mobilization and both normal immature and leukemic human hematopoietic cells as well as regulation of hematopoiesis and engraftment potential.     

Reversible cell surface expression of CD38 on CD34-positive human hematopoietic repopulating cells

OBJECTIVE: Although increased expression of CD38 on the surface of human CD34(+) cells is associated with differentiation, we reported recently that both lineage-negative (Lin(-)) CD34(+)CD38(-) and Lin(-)CD34(+)CD38(lo) fractions of cord blood contain primitive severe combined immunodeficient (SCID)-repopulating cells (SRC). Thus, it is important to determine if a hierarchical relationship exists between the SRC from these two populations or if CD38 is reversibly expressed. MATERIALS AND METHODS: To determine if SRC from the CD34(+)CD38(-) and CD34(+)CD38(lo) cell fractions could generate SRC of the same and/or alternate CD38 expression, cells from primary nonobese diabetic/SCID mice transplanted with CD34(+)CD38(-) cells were resorted into both CD34(+)CD38(-) and CD34(+)CD38(lo) fractions and injected into separate secondary recipients, which were evaluated for human cell engraftment 7 to 10 weeks later. As primary mice transplanted with CD34(+)CD38(lo) cells also contained cells of both immunophenotype, these cells were also resorted and transplanted into separate secondary recipients. The cell-cycle status of various CD34(+) SRC fractions were evaluated using Hoechst 33342 and Pyronin Y staining in order to determine if CD38 expression was coordinated with divisional activation. RESULTS: Each cell fraction obtained from primary recipients was able to reconstitute secondary mice, indicating that CD38 expression reversibly oscillates between negative and low levels on CD34(+) repopulating cells. CD38 expression on repopulating cells correlated with a transition between the G(0) and G(1) phases of the cell cycle. CONCLUSION: CD38 is reversibly expressed on CD34(+) SRC between negative and low levels and corresponds to a change in the cell-cycle state. These observations establish a foundation to uncover the molecular program of stem cell regulation and underscore the importance of functional assessments when isolating and characterizing human hematopoietic stem cells.     

Donor CD4+CD25+ T cells promote engraftment and tolerance following MHC-mismatched hematopoietic cell transplantation

Allogeneic bone marrow transplantation (BMT) is a potentially curative treatment for both inherited and acquired diseases of the hematopoietic compartment; however, its wider use is limited by the frequent and severe outcome of graft-versus-host disease (GVHD). Unfortunately, efforts to reduce GVHD by removing donor T cells have resulted in poor engraftment and elevated disease recurrence. Alternative cell populations capable of supporting allogeneic hematopoietic stem/progenitor cell engraftment without inducing GVHD could increase numbers of potential recipients while broadening the pool of acceptable donors. Although unfractionated CD4(+) T cells have not been shown to be an efficient facilitating population, CD4(+)CD25(+) regulatory cells (T-reg's) were examined for their capacity to support allogeneic hematopoietic engraftment. In a murine fully major histocompatibility complex (MHC)-mismatched BMT model, cotransplantation of donor B6 T-reg's into sublethally conditioned BALB/c recipients supported significantly greater lineage-committed and multipotential donor progenitors in recipient spleens 1 week after transplantation and significantly increased long-term multilineage donor chimerism. Donor engraftment occurred without GVHD-related weight loss or lethality and was associated with tolerance to donor and host antigens by in vitro and in vivo analyses. Donor CD4(+)CD25(+) T cells may therefore represent a potential alternative to unfractionated T cells for promotion of allogeneic engraftment in clinical hematopoietic cell transplantation.     

Definitive hematopoietic commitment within the embryonic vascular endothelial-cadherin(+) population

OBJECTIVE: The aim of this study was to assess the potential of FLK1(+) and vascular endothelial (VE)-cadherin(+) populations from different stages of embryonic development to generate hematopoietic cells ex vivo and to contribute to the hematopoietic systems of recipient mice. MATERIALS AND METHODS: FLK1(+) of VE-cadherin(+) cells were isolated from 7.5- to 9.5-dpc concepti and cultured ex vivo on OP9 stromal cells and hematopoietic development examined. VE-cadherin(+)CD45(-) cells from 8.5- and 9.5-dpc concepti were injected intrahepatically into newborn busulfan-treated SCID recipients and engraftment monitored. RESULTS: VE-cadherin(+) cells from 7.5- and 8.5-dpc concepti can readily generate hematopoi-etic cells ex vivo compared to FLK1(+) VE-cadherin(-) cells. Similar hematopoietic potential can be found in the VE-cadherin(+) cells from the 8.5-dpc yolk sac. When VE-cadherin(+)CD45(-) cells were injected into SCID recipients, long-term engraftment, particularly within the lymphoid system, was observed. This potential was observed in VE-cadherin(+)CD45(-) cells from 9.5-dpc embryo or yolk sac but from tissues from younger concepti. CONCLUSIONS: FLK1(+)VE-cadherin(-) cells, possibly representing the lateral plate mesoderm, are not as effective at generating hematopoietic cells compared to similarly staged VE-cadherin(+) cells. VE-cadherin(+)CD45(-) cells can also contribute to the hematolymphoid system of intrahepatically injected newborn SCID recipients, suggesting that cells bearing an endothelial phenotype are capable of generating long-term hematopoietic precursors.     

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.     

Human CD34+ cell preparations contain over 100-fold greater NOD/SCID mouse engrafting capacity than do CD34- cell preparations.

OBJECTIVE: The CD34 cell surface marker is used widely for stem/progenitor cell isolation. Since several recent studies reported that CD34(-) cells also have in vivo engrafting capacity, we quantitatively compared the engraftment potential of CD34(+) vs CD34(-) cell preparations from normal human placental/umbilical cord blood (CB), bone marrow (BM), and mobilized peripheral blood (PBSC) specimens, using the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. METHODS: CD34(+) and CD34(-) cell preparations were purified by four different approaches in 14 individual experiments involving 293 transplanted NOD/SCID mice. In most experiments, CD34(+) cells were depleted twice (CD34(=)) in order to obtain efficient depletion of CD34(+) cells from the CD34(-) cell preparations. RESULTS: Dose-dependent levels of human hematopoietic cells were observed after transplantation of CD34(+) cell preparations. To rigorously assess the complementary CD34(-) cell preparations, cell doses 10- to 1000-fold higher than the minimum dose of the CD34(+) cell preparations necessary for engraftment were transplanted. Nevertheless, of 125 NOD/SCID mice transplanted with CD34(-) cell preparations purified from the same starting cells, only six mice had detectable human hematopoiesis, by flow cytometric or PCR assay. CONCLUSIONS: CD34(-) cells provide only a minor contribution to hematopoietic engraftment in this in vivo model system, as compared to CD34(+) cells from the same samples of noncultured human cells. Hematopoiesis derived from actual CD34(-) cells is difficult to distinguish from that due to CD34(+) cells potentially contaminating the preparations.     

Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice

In an attempt to develop efficient procedures of human hematopoietic gene therapy, retrovirally transduced CD34(+) cord blood cells were transplanted into NOD/SCID mice to evaluate the repopulating potential of transduced grafts. Samples were prestimulated on Retronectin-coated dishes and infected with gibbon ape leukemia virus (GALV)-pseudotyped FMEV vectors encoding the enhanced green fluorescent protein (EGFP). Periodic analyses of bone marrow (BM) from transplanted recipients revealed a sustained engraftment of human hematopoietic cells expressing the EGFP transgene. On average, 33.6% of human CD45(+) cells expressed the transgene 90 to 120 days after transplantation. Moreover, 11.9% of total NOD/SCID BM consisted of human CD45(+) cells expressing the EGFP transgene at this time. The transplantation of purified EGFP(+) cells increased the proportion of CD45(+) cells positive for EGFP expression to 57. 7% at 90 to 120 days after transplantation. At this time, 18.9% and 4.3% of NOD/SCID BM consisted of CD45(+)/EGFP(+) and CD34(+)/EGFP(+) cells, respectively. Interestingly, the transplantation of EGFP(-) cells purified at 24 hours after infection also generated a significant engraftment of CD45(+)/EGFP(+) and CD34(+)/EGFP(+) cells, suggesting that a number of transduced repopulating cells did not express the transgene at that time. Molecular analysis of NOD/SCID BM confirmed the high levels of engraftment of human transduced cells deduced from FACS analysis. Finally, the analysis of the provirus insertion sites by conventional Southern blotting indicated that the human hematopoiesis in the NOD/SCID BM was predominantly oligoclonal.     

Engraftment potential of human fetal hematopoietic cells in NOD/SCID mice is not restricted to mitotically quiescent cells

During fetal development, there is a continued demand for large numbers of primitive and mature hematopoietic cells. This demand may require that all potential hematopoietic stem cells (HSCs) migrate effectively to emerging hematopoietic sites and subsequently contribute to blood cell production, regardless of their cell cycle status. We recently established that umbilical cord blood cells in the G(1) phase of the cell cycle have a repopulating potential similar to cells in G(0), suggesting that cycling prenatal and neonatal HSCs may have the same functional capabilities described for quiescent, but not cycling, cells from adult sources. To establish the relationship between cell cycle status and hematopoietic potential at early stages of human ontogeny, the in vivo engraftment potential of mitotically defined fetal liver (FL) and fetal bone marrow (FBM) cells were examined in NOD/SCID recipients. Following transplantation of the same numbers of G(0), G(1), or S/G(2)+M CD34(+) cells from FL, equivalent percentages of recipient mice were chimeric (55%, 60%, and 60%, respectively). FBM-derived CD34(+) cells in all phases of the cell cycle engrafted in conditioned recipients and sustained human hematopoiesis, albeit at lower levels than their FL-derived counterparts. Multilineage differentiation was evident in all transplanted mice independent of the source or cell cycle status of graft cells. In addition, levels of chimerism in mice transplanted with fetal blood-derived G(0) or G(1) CD34(+) lineage-depleted cells were similar. These results support the assertion that mitotically quiescent and cycling fetal hematopoietic cells contain marrow-repopulating stem cells capable of multilineage engraftment in NOD/SCID mouse recipients.     

CD34 expression by murine hematopoietic stem cells mobilized by granulocyte colony-stimulating factor

Controversy has existed about CD34 expression by hematopoietic stem cells. We recently reported that CD34 expression reflects the activation state of stem cells by using a murine transplantation model. It has been generally held that mobilized blood stem cells express CD34.However, it has also been reported that mobilized stem cells and progenitors are in G0/G1 phases of the cell cycle. To address the state of CD34 expression by the mobilized stem cells, we again used the mouse transplantation model. We prepared CD34(-) and CD34(+) populations of nucleated blood cells from granulocyte colony-stimulating factor-treated Ly-5.1 mice and assayed each population for long-term engrafting cells in lethally irradiated Ly-5.2 mice. The majority of the stem cells were in the CD34(+) population. The CD34 expression by mobilized stem cells was reversible because re-transplantation of Ly-5.1 CD34(-) marrow cells harvested from the Ly-5.2 recipients of CD34(+)-mobilized stem cells 8 months posttransplantation revealed long-term engraftment. These results may support the use of total CD34(+) cells in mobilized blood as a predictor for engraftment and CD34 selection for enrichment of human stem cells. (Blood. 2000;96:1989-1993)     

Similar repopulating capacity of mitotically active and resting umbilical cord blood CD34(+) cells in NOD/SCID mice

It was hypothesized that during mammalian development, the extensive need for hematopoietic cells requires equal contribution to blood cell production from both quiescent and cycling hematopoietic stem cells (HSCs) while maintaining the stem cell pool. To investigate this hypothesis, the engraftment potential of umbilical cord blood (UCB) CD34(+) cells residing in either G(0) (G(0)CD34(+) cells) or G(1) (G(1)CD34(+) cells) phases of the cell cycle was assessed in nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice. Whereas the level of chimerism in mice transplanted with UCB G(0)CD34(+) cells was 69.9% +/- 24.0%, mice receiving equal numbers of G(1)CD34(+) cells harbored 46.7% +/- 21.3% human cells 8 weeks posttransplantation. Both groups of cells sustained multilineage differentiation and the production of CD34(+) cells in recipient animals. The relationship between the number of transplanted G(0)CD34(+) or G(1)CD34(+) cells and the level of chimerism was analyzed by a general linear models procedure. Although the initial level of chimerism following transplantation of G(0)CD34(+) cells was higher than that sustained by G(1)CD34(+) cells, the increment in the degree of chimerism obtained with each additional 10(3) cells of either phenotype was identical, suggesting that the reconstitution potential of these 2 types of cells was similar. Of interest is that human cells recovered from primary recipients of both G(0)CD34(+) and G(1)CD34(+) cells engrafted in secondary NOD/SCID recipients, albeit at a substantially lower level, confirming the primitive nature of UCB CD34(+) cells residing in G(1).     

Homing and engraftment potential of Sca-1(+)lin(-) cells fractionated on the basis of adhesion molecule expression and position in cell cycle

Engraftment potential of hematopoietic stem cells (HSCs) is likely to be dependent on several factors including expression of certain adhesion molecules (AMs) and degree of mitotic quiescence. The authors investigated the functional properties and engraftment potential of Sca-1(+)lin(-) cells subfractionated on the basis of expression, or lack thereof, of CD11a, CD43, CD49d, CD49e, or CD62L and correlated that expression with cell cycle status and proliferative potential of engrafting fractions. Donor-derived chimerism in mice receiving CD49e(+) or CD43(+) Sca-1(+)lin(-) cells was greater than that in mice receiving cells lacking these 2 markers, while Sca-1(+)lin(-) cells positive for CD11a and CD62L and bright for CD49d expression mediated minimal engraftment. AM phenotypes enriched for engraftment potential contained the majority of high proliferative potential-colony forming cells, low proliferative potential-colony forming cells, and cells providing rapid in vitro expansion. Cell cycle analysis of AM subpopulations revealed that, regardless of their bone marrow repopulating potential, Sca-1(+)lin(-) AM(-) cells contained a higher percentage of cells in G(0)/G(1) than their AM(+) counterparts. Interestingly, engrafting phenotypes, regardless of the status of their AM expression, were quicker to exit G(0)/G(1) following in vitro cytokine stimulation than their opposing phenotypes. When engrafting phenotypes of Sca-1(+)lin(-) AM(+) or AM(-) cells were further fractionated by Hoechst 33342 into G(0)/G(1) or S/G(2)+M, cells providing long-term engraftment were predominantly contained within the quiescent fraction. These results define a theoretical phenotype of a Sca-1(+)lin(-) engrafting cell as one that is mitotically quiescent, CD43(+), CD49e(+), CD11a(-), CD49d(dim), and CD62L(-). Furthermore, these data suggest that kinetics of in vitro proliferation may be a good predictor of engraftment potential of candidate populations of HSCs. (Blood. 2000;96:1380-1387)     

Prolonged ex vivo culture of cord blood CD34(+) cells facilitates myeloid and megakaryocytic engraftment in the non-obese diabetic severe combined immunodeficient mouse model

A clinical goal for ex vivo expansion of cord blood (CB) CD34(+) cells is to shorten the period of neutropenia and thrombocytopenia following myeloablative therapy and transplantation. Prolongation of cytokine expansion leads to the production of greater numbers of cells, and should have an impact on neutrophil and platelet recovery. Furthermore, expansion of CD34(+) cells should support the continued production of neutrophils and platelets in the 6-week period following transplantation. We tested these hypotheses by characterization of the kinetics (human CD45(+) cells in the blood) and phenotype (CD45, CD34, CD61, CD33, CD19 and CD3) of human engraftment in the non-obese diabetic severe combined immunodeficient mouse (NOD-SCID) following 7 or 14 d of ex vivo expansion of CB CD34(+) cells. Mice transplanted with 14 d cells showed greater percentages of human CD45(+) cells in the blood, bone marrow and spleen than mice transplanted with unexpanded cells or 7 d cells. Prolonging cytokine exposure of CD34(+) cells and transplantation with increasing numbers of input cells facilitated the production of absolute numbers of CD34(+), CD33(+), CD61(+) and CD19(+) cells in vivo. Furthermore, analysis of SCID engrafting potential showed that prolongation of culture duration facilitates in vivo expansion of CD45(+), CD34(+) and CD19(+) cells after transplantation. It is anticipated that prolonged (2 weeks) ex vivo culture of CB will have a beneficial clinical effect.     

A Stro-1(+) human universal stromal feeder layer to expand/maintain human bone marrow hematopoietic stem/progenitor cells in a serum-free culture system

OBJECTIVE: To compare the ability of allogeneic versus autologous purified human Stro-1(+) mesenchymal stem cell (MSC) populations from different human donors to support the ex vivo expansion and maintenance of human hematopoietic stem/progenitor cells (HSCs). Furthermore, we compared the results obtained with MSC as a feeder layer to traditional allogeneic stromal layers grown in long-term bone marrow culture media (LT-ST). METHODS: Adult human bone marrow CD34(+)-enriched cells were cultured in serum-free medium for 2 to 3 weeks over the respective MSC-irradiated feeder layers or over traditional allogeneic LT- ST stromal layers in the presence of stem cell factor, basic fibroblast growth factor, leukemia inhibitory factor, and Flt-3 and analyzed every 2 to 4 days for expansion, phenotype, and clonogenic ability. RESULTS: There was a progressive expansion of total numbers of cells in all the experimental groups; however, allogeneic MSCs were more efficient at expanding CD34(+)CD38(-) cells and showed a higher clonogenic potential than both allogeneic LT-ST and autologous MSCs. The differentiative potential of cells cultured on both MSC and LT-ST was primarily shifted toward myeloid lineage; however, only MSCs were able to maintain/expand a CD7(+) population with lymphocytic potential. Importantly, transplantation into preimmune fetal sheep demonstrated that the HSCs cultured over MSCs retained their engraftment capability. CONCLUSION: These results indicate that purified Stro-1(+) MSCs may be used as a universal and reproducible stromal feeder layer to efficiently expand and maintain human bone marrow HSCs ex vivo.     

Expression of Fas and Fas-ligand in donor hematopoietic stem and progenitor cells is dissociated from the sensitivity to apoptosis

OBJECTIVE: The interaction between the Fas receptor and its cognate ligand (FasL) has been implicated in the mutual suppression of donor and host hematopoietic cells after transplantation. Following the observation of deficient early engraftment of Fas and FasL-defective donor cells and recipients, we determined the role of the Fas-FasL interaction. METHODS: Donor cells were recovered after syngeneic (CD45.1-->CD45.2) transplants from various organs and assessed for expression of Fas/FasL in reference to lineage markers, carboxyfluorescein succinimidyl ester dilution, Sca-1 and c-kit expression. Na?ve and bone marrow-homed cells were challenged for apoptosis ex vivo. RESULTS: The Fas receptor and ligand were markedly upregulated to 40% to 60% (p < 0.001 vs 5-10% in na?ve cells) within 2 days after syngeneic transplantation, while residual host cells displayed modest and delayed upregulation of these molecules ( approximately 10%). All lin(-)Sca(+)c-kit(+) cells were Fas(+)FasL(+), including 95% of Sca-1(+) and 30% of c-kit(+) cells. Fas and FasL expression varied in donor cells that homed to bone marrow, spleen, liver and lung, and was induced by interaction with the stroma, irradiation, cell cycling, and differentiation. Bone marrow-homed donor cells challenged with supralethal doses of FasL were insensitive to apoptosis (3.2% +/- 1% vs 38% +/- 5% in na?ve bone marrow cells), and engraftment was not affected by pretransplantation exposure of donor cells to an apoptotic challenge with FasL. CONCLUSION: There was no evidence of Fas-mediated suppression of donor and host cell activity after transplantation. Resistance to Fas-mediated apoptosis evolves as a functional characteristic of hematopoietic reconstituting stem and progenitor cells, providing them competitive engraftment advantage over committed progenitors.     

Kinetics of engraftment of CD34(-) and CD34(+) cells from mobilized blood differs from that of CD34(-) and CD34(+) cells from bone marrow

OBJECTIVE: Mobilized peripheral blood (PB) progenitors are increasingly used in autologous and allogeneic transplantation. However, the short- and long-term engraftment potential of mobilized PB or bone marrow (BM) has not been directly compared. Although several studies showed that BM-derived Lin(-)CD34(-) cells contain hemopoietic progenitors, no studies have addressed whether Lin(-)CD34(-) cells from mobilized PB contain hemopoietic progenitors. Here, we compared the short- and long-term engraftment potential of CD34(+) cells and Lin(-)CD34(-) cells in BM and PB of normal donors who received 5 days of granulocyte colony-stimulating factor (G-CSF). MATERIALS AND METHODS: 35 x 10(3) CD34(+) or Lin(-)CD34(-) cells from G-CSF mobilized BM and PB of normal donors were transplanted in 60-day-old fetal sheep. Animals were evaluated 2 and 6 months after transplantation for human hemopoietic cells. In addition, cells recovered after 2 months from fetal sheep were serially passaged to secondary and tertiary recipients to assess long-term engrafting cells. RESULTS: Mobilized PB CD34(+) cells supported earlier development of human hemopoiesis than BM CD34(+) cells. When serially transferred to secondary and tertiary recipients, earlier exhaustion of human hematopoiesis was seen for PB than BM CD34(+) cells. A similar degree of chimerism was seen for Lin(-)CD34(-) cells from PB or BM in primary recipients. We again observed earlier exhaustion of human hemopoiesis with serial transplantation of PB than BM Lin(-)CD34(-) cells. CONCLUSIONS: Differences exist in the short- and long-term repopulating ability of cells in PB and BM from G-CSF mobilized normal donors, and this is independent of the phenotype. Studies are ongoing to examine if this reflects intrinsic differences in the repopulating potential between progenitors from PB and BM, or a lower frequency of long-term repopulating cells in PB than BM CD34(+) and Lin(-)CD34(-) cells, that may not be apparent if larger numbers of cells are transplanted.