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.     

Differentiative potential of human metanephric mesenchymal cells

OBJECTIVE: To evaluate the ability of mesenchymal cells derived from nonhematopoietic organs to form blood and other tissues in vitro and in vivo. MATERIALS AND METHODS: Because of its mesodermic derivation, human fetal kidney was used as a source of mesenchymal cells. Two populations of kidney cells were studied at a nonclonal level: a crude preparation, and an adherent fraction that was derived from the first by propagation in vitro (MNMC). Both populations were transplanted into sheep fetuses and analyzed at intervals for the presence of human cells in different organs by flow cytometry, PCR, immunohistochemistry, and in situ hybridization. Secondary transplantation studies were performed using human hematopoietic cells obtained from the bone marrow (BM) of primary recipients. RESULTS: MNMC were Thy-1(+), CD51(+), CD44(+), CD45(-), and vimentin(+), a phenotype consistent with that of metanephric mesenchyme. The crude population displayed the same phenotype but was contaminated with 0.4% CD34(+)CD45(+) cells. Cells with hepatocyte-like morphology and phenotype were obtained from the MNMC after culture in specific inducing media. After transplantation, both populations of cells produced multilineage hematopoietic engraftment and gave rise to CD34(+) cells. Successful hematopoietic engraftment in secondary recipients demonstrated the generation of long-term engrafting hematopoietic stem cells from MNMC. PCR analysis confirmed human hematopoietic engraftment and revealed that human cells were also present within other organs. Liver sections of transplanted animals contained human albumin-producing hepatocyte-like cells. CONCLUSION: A human metanephric mesenchymal cell population simultaneously gave rise to human blood and liver-like cells, suggesting that mesenchymal cells may represent a broad population of putative stem cells in multiple adult organs.     

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.     

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.     

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.     

Human embryonic stem cell-derived hematopoietic cells are capable of engrafting primary as well as secondary fetal sheep recipients

The human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and postnatal human sources. CD34+/lineage- or CD34+/CD38- cells isolated from human embryonic stem cells (hESCs) differentiated on S17 feeder layer were transplanted by intraperitoneal injections into fetal sheep. Chimerism in primary transplants was established with polymerase chain reaction (PCR) and flow cytometry of bone marrow and peripheral blood samples. Whole bone marrow cells harvested from a primary recipient were transplanted into a secondary recipient. Chimerism was established as described before. This animal was stimulated with human GM-CSF, and an increase in human hematopoietic activity was noted by flow cytometry. Bone marrow aspirations cultured in methylcellulose generated colonies identified by PCR to be of human origin. We therefore conclude that hESCs are capable of generating hematopoietic cells that engraft primary recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the secondary recipient.     

CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.     

Ex vivo expanded cord blood cells provide rapid engraftment in fetal sheep but lack long-term engrafting potential

OBJECTIVE: Cord blood (CB) products are becoming routinely used in unrelated allogeneic transplantation for smaller pediatric patients. Because of the low numbers of cells in CB compared to bone marrow or peripheral blood progenitor cells, their use is more limited in larger adults. Therefore, we developed ex vivo expansion conditions for CB and currently are transplanting ex vivo expanded CB products to patients receiving high-dose chemotherapy. As there is concern that ex vivo expansion may exhaust long-term engrafting cells, the current clinical protocols consist of both an expanded fraction and an unexpanded fraction. To determine the effect of expansion culture on long-term engrafting cells, we evaluated the short- and long-term engrafting potential of ex vivo expanded CB using a fetal sheep xenogeneic transplant model. MATERIAL AND METHODS: CD 34(+) cells were selected from CB products and cultured in a two-step procedure in the presence of stem cell factor, megakaryocyte growth and differentiation factor, and granulocyte colony-stimulating factor for 14 days. Starting cells (CD34(+) cells), and cultured cells (day 7 and day 14 cells) were transplanted in 60-day-old fetal sheep and evaluated at various time points post transplant for the presence of human cells. Long-term engrafting cells were assessed by serial passage into secondary and tertiary recipients. RESULTS: Day 14 expanded CB cells provided more rapid engraftment than either the day 7 expanded cells or the day 0 cells; however, this engraftment was transient, and no human cells were detectable at 16 months post transplant in the animals that received the day 14 expanded cells. Day 0 cells had engrafted animals at 2 months post transplant and both the day 0 and day 7 cells persisted to 16 months or longer. In the secondary animals, the day 0 and day 7 cells engrafted equivalently at 3 months post transplant; however, no secondary engraftment resulted from the day 14 cells. The levels of engraftment in secondary animals receiving day 7 cells decreased with time to barely detectable levels at 12 months post transplant. CONCLUSIONS: Ex vivo expansion of CB CD34(+) cells under the conditions described results in the generation of increased mature cells and progenitors that are capable of more rapid engraftment in fetal sheep compared to unexpanded CB CD34(+) cells. The expanded cells engrafted primary sheep but lacked secondary and tertiary engrafting potential. These studies demonstrate that although ex vivo expanded cells may be able to provide rapid short-term engraftment, the long-term potential of expanded grafts may be compromised. Therefore, clinical protocols may require transplantation of two fractions of cells, an expanded CB graft to provide rapid short-term engraftment and an unmanipulated fraction of CB graft to provide stem cells for long-term engraftment.     

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 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.     

Stem cell factor improves SCID-repopulating activity of human umbilical cord blood-derived hematopoietic stem/progenitor cells in xenotransplanted NOD/SCID mouse model

Poor in vivo homing capacity of hematopoietic stem/progenitor cells (HS/PCs) from umbilical cord blood (UCB) can be reversed by short-term ex vivo manipulation with recombinant human stem cell factor (rHuSCF). This study was designed to evaluate the effect of ex vivo manipulation of UCB-derived HS/PCs with rHuSCF on human cell engraftment rates in xenotransplanted NOD/SCID mouse model. The human cell engraftment rates in xenotransplanted primary and secondary NOD/SCID mice were characterized using four-color flow cytometric analysis and progenitor assay. Grafts of rHuSCF-treated UCB CD34(+) cells resulted in significantly higher levels of human cell engraftment than that of nontreated ones in both xenotransplanted primary and secondary NOD/SCID recipients. Fresh UCB CD34(+) cells did not express either of the matrix metalloproteinase (MMP) family members MMP-2 or MMP-9. rHuSCF-treated UCB CD34(+) cells expressed significant levels of MMP-2 and MMP-9. Pretreatment of UCB CD34(+) cells with the specific MMP inhibitor completely blocked human cell engraftment in xenotransplanted NOD/SCID recipients. Our results indicate that ex vivo manipulation of human HS/PCs with rHuSCF might provide an optimal approach to develop more effective stem cell-based therapies in situations where engraftment is delayed due to limiting HS/PCs number, for example, UCB transplantation.     

Differential long-term and multilineage engraftment potential from subfractions of human CD34+ cord blood cells transplanted into NOD/SCID mice

Over the past decade xenotransplantation systems have been used with increasing success to gain a better understanding of human cells that are able to initiate and maintain the hematopoietic system in vivo. The nonobese diabetic/severe combined immunodeficiency (SCID) mouse has been a particularly useful model. Human cells capable of hematopoietic repopulation in this mouse, termed SCID-repopulating cells, have been assumed to represent the most primitive elements of the hematopoietic system, responsible for long-term maintenance of hematopoiesis. However, we demonstrate that SCID-repopulating cells present in the CD34(+) cell fraction of cord blood can be segregated into subpopulations with distinct repopulation characteristics. CD34(+)/CD38(+) progenitors can repopulate recipients rapidly, but can only maintain the graft for 12 weeks or less and have no secondary repopulation potential. Conversely, the more primitive CD34(+)/CD38(-) subpopulation repopulates recipients more gradually, can maintain the graft for at least 20 weeks, and contains cells with serial repopulation potential throughout the engraftment period. Additionally, a much higher frequency of T cell precursors are found among SCID-repopulating cells in the CD34(+)/CD38(-) subpopulation. These findings demonstrate that cells with variable repopulation potential comprise the human CD34(+) population and that short- and long-term potential of human precursors can be evaluated in the mouse model.     

Engraftment of human hematopoietic precursor cells with secondary transfer potential in SCID-hu mice

Human fetal bone fragments implanted subcutaneously in immunodeficient (SCID) mice maintain active human hematopoiesis. In this study, we show that this human hematopoietic microenvironment supports the engraftment and differentiation of HLA-mismatched, CD34+ primitive hematopoietic progenitor cells isolated from fetal and adult human bone marrow (BM). The BM CD34+ cells were depleted of CD2, CD14, CD15, CD16, glycophorin A, and CD19 lineage-committed cells (CD34+Lin-). Donor cell engraftment was manifested by the presence of B (CD19+) and myeloid (CD33+) cells of donor HLA phenotype. Successful engraftment was observed as early as 4 weeks after fetal BM donor cell injection and sustained for at least 12 weeks, with engraftment success rates of 100% (11/11 grafts) and 92% (11/12 grafts) at 8 and 12 weeks, respectively. Mixed BM chimerism of donor and endogenous cells was consistently observed in SCID-hu bones successfully engrafted with HLA-mismatched CD34+Lin- donor cells. Preconditioning of the SCID-hu bone with a single dose of sublethal (350 rad) whole body irradiation (WBI) immediately before cell injection enhanced the repopulation of the bone grafts with donor cells and, in some instances, resulted in complete repopulation. After WBI, as few as 500 fetal bone marrow CD34+Lin- cells injected in the human bone grafts resulted in donor-derived hematopoiesis. Donor progenitor cells recovered from the SCID-hu bone grafts 8 weeks postinjection had the capacity to repopulate secondary groups of HLA-disparate fetal human bones in SCID-hu mice with B and myeloid cells as well as CD34+ cells in some recipients. In addition, these cells repopulated fetal human thymus fragments in SCID mice with donor thymocytes including immature CD4+CD8+ and mature CD4+CD8- as well as CD4-CD8+ subsets. These results indicate that the fetal human bone implants of SCID-hu mice can support the maintenance of a cell population that has both multilineage potential and repopulating potential for periods of time as long as 16 weeks. The SCID-hu bone model consistently supported the engraftment of both fetal and adult CD34+Lin- cells without the administration of exogenous human cytokines to these animals. This model is currently being used to permit the isolation and characterization of candidate human hematopoietic stem cells (HSCs) and provide important information critical for human HSC therapy in humans.     

Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system

This report describes stroma-based and stroma-free cultures that maintain long-term engrafting hematopoietic cells for at least 14 days ex vivo. Umbilical cord blood (UCB) CD34(+) cells were cultured in transwells above AFT024 feeders with fetal-liver-tyrosine-kinase (FL) + stem cell factor (SCF) + interleukin 7 (IL-7), or FL + thrombopoietin (Tpo). CD34(+) progeny were transplanted into nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice or preimmune fetal sheep. SCID repopulating cells (SRC) with multilineage differentiation potential were maintained in FL-SCF-IL-7 or FL-Tpo containing cultures for up to 28 days. Marrow from mice highly engrafted with uncultured or expanded cells induced multilineage human hematopoiesis in 50% of secondary but not tertiary recipients. Day 7 expanded cells engrafted primary, secondary, and tertiary fetal sheep. Day 14 expanded cells, although engrafting primary and to a lesser degree secondary fetal sheep, failed to engraft tertiary recipients. SRC that can be transferred to secondary recipients were maintained for at least 14 days in medium containing glycosaminoglycans and cytokines found in stromal supernatants. This is the first demonstration that ex vivo culture in stroma-noncontact and stroma-free cultures maintains "long-term" engrafting cells, defined by their capacity to engraft secondary or tertiary hosts. (Blood. 2001;97:3441-3449)     

In utero transplantation of human fetal haemopoietic cells in NOD/SCID mice

We have previously demonstrated that high levels of allogeneic, donor-derived mouse haemopoietic progenitor cells engraft following in utero transplantation in NOD/SCID mice. To evaluate whether the fetal NOD/SCID haemopoietic microenvironment supports the growth and development of human fetal haemopoietic progenitor cells, we injected fetal liver mononuclear cells (FL) or fetal bone marrow (FBM) derived CD34⁺ cells into NOD/SCID mice on day 13/14 of gestation. At 8 weeks of age 12% of FBM recipients and 10% of FL recipients were found to have been successfully engrafted with CD45⁺ human cells. CD45⁺ cells were present in the BM of all chimaeric animals; 5/6 recipients showed engraftment of the spleen, and 4/6 recipients had circulating human cells in the peripheral blood (PB). The highest levels of donor cells were found in the BM, with up to 15% of the nucleated cells expressing human specific antigens. Multilineage human haemopoietic engraftment, including B cells (CD19), myelomonocytic cells (CD13/33) and haemopoietic progenitor cells (CD34), was detected in the BM of chimaeric mice. In contrast, no human CD3⁺ cells were detected in any of the tissues evaluated. When the absolute number of engrafted human cells in the PB, BM and spleens of chimaeric mice was determined, a mean 16-fold expansion of human donor cells was observed. Although multilineage engraftment occurs in these fetal recipients, both the frequency and the levels of engraftment are lower than those previously reported when human cells are transplanted into adult NOD/SCID recipients.     

In vivo haematopoietic potential of human neural stem cells

The fetal sheep model was used to compare the in vivo haematopoietic potential of human neural stem cells (NSC) versus bone marrow (BM)-derived haematopoietic stem cells (HSC). To this end, sheep were transplanted with either 8 x 10(5) NSC (n = 11) or HSC, CD34(+)Lin(-) (n = 5), and subsequently analysed for haematopoietic chimaerism. While HSC-transplanted sheep displayed robust donor-derived haematopoiesis starting at less than 2 months post-transplant, NSC recipients exhibited haematopoietic engraftment at much later time points. Nevertheless, chimaerism persisted in both groups throughout the course of this study. Transplantation of secondary recipients with human CD45(+)/HLA-DR(+) cells from the BM of NSC primary recipients at 14 and 16 months post-transplant demonstrated that long-term engrafting HSC were present in these animals. At 6 months post-transplant, both NSC- and HSC-transplanted sheep were mobilised with granulocyte colony-stimulating factor. In contrast to HSC-transplanted animals, levels of human blood cells in peripheral blood of NSC-transplanted sheep remained low throughout mobilisation. Our results show that, although human NSC were able to give rise to multilineage haematopoiesis in our model, the levels, timing of blood cell production and the ability to respond to cytokine mobilisation were different, suggesting that human NSCs latent haematopoietic potential is inherently different from that of true HSC.     

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.     

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.     

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.     

Engraftment potential of different sources of human hematopoietic progenitor cells in BNX Mice

Human hematopoietic progenitor cells (HPCs) from mobilized peripheral blood mononuclear cells (PBMCs), adult bone marrow (ABM), and fetal bone marrow (FBM) were evaluated for their ability to produce multilineage human hematopoietic engraftment in vivo. Sublethally irradiated BNX (beige/nude/xid) mice were injected with either unfractionated cells or CD34+ cells purified from these sources. The presence of human cells in the mouse PB, BM, and spleen was evaluated by flow cytometry at either 6 to 8 weeks or 6 months postinjection. Recipients with > or = 1% human cells in any of these tissues were considered chimeric. Of 26 mice injected with FBM, 4 showed up to 73% human cells in the BM or spleen at 6 months. The phenotypes of these cells included CD13/33+ myelomonocytic cells (38%), CD19+ B cells (67%), and CD34+ progenitor cells (28%). In contrast, ABM gave rise to a mean of 5% human cells in the PB in 2 of 42 (4%) recipients at 6 to 8 weeks. These circulating human cells were predominantly CD3+, whereas CD13/33+ and CD34+ cells were detected in the BM for up to 6 months. A total of 18% of mice injected with PBMCs showed a mean of 36% human cells in the PB. Both the BM and spleens of PBMC-injected mice contained CD3+ cells in a proportion similar to that observed in the PB. These CD3+ cells were phenotypically mature CD4+,CD8- or CD4-,CD8+ T cells and coexpressed a variety of Vbeta T-cell receptor (TCR) genes. The percentage of CD3+ cells in the circulation of chimeric recipients injected with either FBM, ABM, or PBMCs correlated well with the input CD3+ cell dose for each of these HPC sources (r = .99). The high levels of engraftment of CD3+ cells in recipients of PBMCs and the long-term multilineage engraftment of FBM recipients have important implications for developing strategies to study the regulation of these human cells in vivo.