Temporal definition of haematopoietic stem cell niches in a large animal model of in utero stem cell transplantation.

The fetal sheep model has served as a biologically relevant and translational model to study in utero haematopoietic stem cell transplantation (IUHSCT), yet little is known about the ontogeny of the bone marrow (BM) niches in this model. Because the BMmicroenvironment plays a critical role in the outcome of haematopoietic engraftment, we have established the correlation between the fetal-sheep and fetal-human BM niche ontogeny, so that studies addressing the role of niche development at the time of IUHSCT could be accurately performed. Immunofluorescence confocal microscopic analysis of sheep fetal bone from gestational days (gd) 25-68 showed that the BM microenvironment commences development with formation of the vascular niche between 25 and 36 gd in sheep; correlating with the events at 10-11 gestational weeks (gw) in humans. Subsequently, between 45 and 51 gd in sheep (c. 14 gw in humans), the osteoblastic/endosteal niche started developing, the presence of CD34(+)  CD45(+) cells were promptly detected, and their number increased with gestational age. IUHSCT, performed in sheep at 45 and 65 gd, showed significant haematopoietic engraftment only at the later time point, indicating that a fully functional BM microenvironment improved engraftment. These studies show that sheep niche ontogeny closely parallels human, validating this model for investigating niche influence/manipulation in IUHSCT engraftment.

Influence of a dual-injection regimen, plerixafor and CXCR4 on in utero hematopoietic stem cell transplantation and engraftment with use of the sheep model.

BACKGROUND AIMS: Inadequate engraftment of hematopoietic stem cells (HSCs) after in utero HSC transplantation (IUHSCT) remains a major obstacle for the prenatal correction of numerous hereditary disorders. HSCs express CXCR4 receptors that allow homing and engraftment in response to stromal-derived factor 1 (SDF-1) ligand present in the bone marrow stromal niche. Plerixafor, a mobilization drug, works through the interruption of the CXCR4-SDF-1 axis. METHODS: We used the fetal sheep large-animal model to test our hypotheses that (i) by administering plerixafor in utero before performing IUHSCT to release fetal HSCs and thus vacating recipient HSC niches, (ii) by using human mesenchymal stromal/stem cells (MSCs) to immunomodulate and humanize the fetal BM niches and (iii) by increasing the CXCR4(+) fraction of CD34(+) HSCs, we could improve engraftment. Human cord blood-derived CD34(+) cells and human bone marrow-derived MSCs were used for these studies. RESULTS: When MSCs were transplanted 1 week before CD34(+) cells with plerixafor treatment, we observed 2.80% donor hematopoietic engraftment. Combination of this regimen with additional CD34(+) cells at the time of MSC infusion increased engraftment levels to 8.77%. Next, increasing the fraction of CXCR4(+) cells in the CD34(+) population albeit transplanting at a late gestation age was not beneficial. Our results show engraftment of both lymphoid and myeloid lineages. CONCLUSIONS: Prior MSC and HSC cotransplantation followed by manipulation of the CXCR4-SDF-1 axis in IUHSCT provides an innovative conceptual approach for conferring competitive advantage to donor HSCs. Our novel approach could provide a clinically relevant approach for enhancing engraftment early in the fetus.

EphB2 isolates a human marrow stromal cell subpopulation with enhanced ability to contribute to the resident intestinal cellular pool.

To identify human bone marrow stromal cell (BMSC) subsets with enhanced ability to engraft/contribute to the resident intestinal cellular pool, we transplanted clonally derived BMSCs into fetal sheep. Analysis at 75 d post-transplantation showed 2 of the 6 clones engrafting the intestine at 4- to 5-fold higher levels (5.03±0.089 and 5.04±0.15%, respectively) than the other clones (P<0.01), correlating with the percentage of donor-derived Musashi-1(+) (12.01-14.17 vs. 1.2-3.8%; P<0.01) or leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5)(+) cells within the intestinal stem cell (ISC) region. Phenotypic and transcriptome analysis determined that the clones with enhanced intestinal contribution expressed high levels of Ephrin type B receptor 2 (EphB2). Intestinal explants demonstrated proliferation of the engrafted cells and ability to generate crypt-like structures in vitro still expressing EphB2. Additional transplants based on BMSC EphB2 expression demonstrated that, at 7 d post-transplant, the EphB2(high) BMSCs engrafted in the ISC region at levels of 2.1 ± 0.2%, while control EphB2(low) BMSCs engrafted at 0.3 ± 0.1% (P<0.01). Therefore we identified a marker for isolating and culturing an expandable subpopulation of BMSCs with enhanced intestinal homing and contribution to the ISC region.

Mesenchymal stem cells contribute to endogenous FVIII:c production.

Besides the liver, it has been difficult to identify which organ(s) and/or cellular component(s) contribute significantly to the production of human FVIII:c (FVIII). Thus far, only endothelial cells have been shown to constitute a robust extrahepatic source of FVIII, possibly explaining both the diverse presence of FVIII mRNA in the body, and the observed increase in FVIII levels during liver failure. Here, we investigate whether human mesenchymal stem cells (MSC), ubiquitously present in different organs, could also contribute to FVIII production. MSC isolated from human lung, liver, brain, and bone marrow expressed FVIII message as determined by quantitative-RT-PCR. Using an antibody specific for FVIII, confocal microscopy, and umbilical cord-derived endothelial cells (HUVEC) as a negative control, we demonstrated that, in MSC, FVIII protein was not stored in granules; rather, it localized to the perinuclear region. Furthermore, functional FVIII was detected in MSC supernatants and cell lysates by aPTT and chromogenic assays. These results demonstrate that MSC can contribute at low levels to the functional FVIII pool, and advance the understanding of the physiology of FVIII production and secretion.

Distinct contribution of human cord blood-derived endothelial colony forming cells to liver and gut in a fetal sheep model.

UNLABELLED: Although the vasculogenic potential of circulating and cord blood (CB)-derived endothelial colony-forming cells (ECFC) has been demonstrated in vitro and in vivo, little is known about the inherent biologic ability of these cells to home to different organs and contribute to tissue-specific cell populations. Here we used a fetal sheep model of in utero transplantation to investigate and compare the intrinsic ability of human CB-derived ECFC to migrate to the liver and to the intestine, and to define ECFC's intrinsic ability to integrate and contribute to the cytoarchitecture of these same organs. ECFCs were transplanted by an intraperitoneal or intrahepatic route (IH) into fetal sheep at concentrations ranging from 1.1-2.6 × 10(6) cells/fetus. Recipients were evaluated at 85 days posttransplant for donor (human) cells using flow cytometry and confocal microscopy. We found that, regardless of the route of injection, and despite the IH delivery of ECFC, the overall liver engraftment was low, but a significant percentage of cells were located in the perivascular regions and retained the expression of hallmark endothelial makers. By contrast, ECFC migrated preferentially to the intestinal crypt region and contributed significantly to the myofibroblast population. Furthermore, ECFC expressing CD133 and CD117 lodged in areas where endogenous cells expressed those same phenotypes. CONCLUSION: ECFC inherently constitute a potential source of cells for the treatment of intestinal diseases, but strategies to increase the numbers of ECFC persisting within the hepatic parenchyma are needed in order to enhance ECFC therapeutic potential for this organ.

A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential.

Here a new, intrinsically pluripotent, CD45-negative population from human cord blood, termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 10(15) cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein, and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model, the preimmune fetal sheep, resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals.

Differences amid bone marrow and cord blood hematopoietic stem/progenitor cell division kinetics.

Human hematopoietic stem/progenitor cells (HSC) isolated based upon specific patterns of CD34 and CD38 expression, despite phenotypically identical, were found to be functionally heterogeneous, raising the possibility that reversible expression of these antigens may occur during cellular activation and/or proliferation. In these studies, we combined PKH67 tracking with CD34/CD38 immunostaining to compare cell division kinetics between human bone marrow (BM) and cord blood (CB)-derived HSC expanded in a serum-free/stromal-based system for 14 days (d), and correlated CD34 and CD38 expression with the cell divisional history. CB cells began dividing 24 h earlier than BM cells, and significantly higher numbers underwent mitosis during the time in culture. By d10, over 55% of the CB-cells reached the ninth generation, whereas BM-cells were mostly distributed between the fifth and seventh generation. By d14, all CB cells had undergone multiple cell divisions, while 0.7-3.8% of BM CD34(+) cells remained quiescent. Furthermore, the percentage of BM cells expressing CD34 decreased from 60.8 +/- 6.3% to 30.6 +/- 6.7% prior to initiating division, suggesting that downmodulation of this antigen occurred before commencement of proliferation. Moreover, with BM, all primitive CD34(+)CD38(-) cells present at the end of culture arose from proliferating CD34(+)CD38(+) cells that downregulated CD38 expression, while in CB, a CD34(+)CD38(-) population was maintained throughout culture. These studies show that BM and CB cells differ significantly in cell division kinetics and expression of CD34 and CD38, and that the inherent modulation of these antigens during ex vivo expansion may lead to erroneous quantification of the stem cell content of the expanded graft.

Immune ontogeny and engraftment receptivity in the sheep fetus.

OBJECTIVE: The biologic explanation for fetal receptivity to donor engraftment and subsequent long-term tolerance following transplantation early in gestation is not known. We investigated the role fetal immune ontogeny might play in fetal transplantation tolerance in sheep. METHODS: Engraftment of allogeneic and xenogeneic HSC was determined 60 days following transplantation at different time points in sheep fetal gestation. Parallel analysis of surface differentiation antigen expression on cells from lymphoid organs of timed gestational age fetal sheep was determined by flow cytometry using available reagents. RESULTS: An engraftment window was identified after day 52 gestation lasting until day 71 (term gestation: 145 days). This period was associated with the expression of the leukocyte common antigen CD45 on all cells in the thymus. Double-positive and single-positive CD4 and CD8 cells began appearing in the thymus just prior (day 45 gestation) to the beginning of the engraftment window, while single-positive CD4 or CD8 cells do not begin appearing in peripheral organs until late in the engraftment period, suggesting deletional mechanisms may be operative. In concert, surface IgM-positive cells express CD45 in the thymus at day 45, with a comparable delay in the appearance of IgM/CD45 cells in the periphery until late in the engraftment window. CONCLUSIONS: These findings support a central role for the thymus in multilineage immune cell maturation during the period of fetal transplantation receptivity. Further, they suggest that fetal engraftment receptivity is due to gestational age-dependent deletional tolerance.

Generation of functional natural killer and dendritic cells in a human stromal-based serum-free culture system designed for cord blood expansion.

OBJECTIVE: We have previously reported on the ability of a mesenchymal stem cell-based serum-free culture system to expand human cord blood (CB) hematopoietic stem cells along the myeloid pathway and simultaneously generate a CD7(+)CD34(-) population. In this study, we investigated the ability of the CD7(+)CD34(-) population to differentiate into natural killer and dendritic cells (DCs). MATERIALS AND METHODS: CB CD34(+) cells were expanded over a mesenchymal stem cell layer in serum-free medium supplemented with stem cell factor, basic fibroblast growth factor, leukemia inhibitor factor, and Flt-3 ligand for 2 weeks. Cultured cells were harvested and CD7(+)CD34(-)Lin(-) cells sorted and plated for 2 additional weeks in either natural killer- or DC-inductive medium. RESULTS: Culture of CD34(+) cells for the first 2 weeks in this system resulted in expansion of the stem cell pool and the myeloid component of the graft, and also produced a 58-fold increase in the CD7(+)CD34(-) cell population. When sorted CD7(+)CD34(-)Lin(-) cells were induced toward a natural killer cell phenotype, further expansion was observed during this time in culture, and differentiation was confirmed by cytotoxic activity and by flow cytometry, with cells displaying CD16 and CD56 in the absence of CD3. Generation of DC cells in culture was also verified by observing both the characteristic dendritic morphology and the dendritic phenotypes HLA-DR(bright)CD123(bright)CD11c(-) and HLA-DR(bright)CD11c(+). CONCLUSION: These results demonstrate the ability of an ex vivo culture system to drive expansion of human CB hematopoietic stem cells, while promoting the immune maturation of the graft and generation of DC and natural killer cells that could then be utilized for adoptive cancer cellular immunotherapy.

Development and characterization of a novel CD34 monoclonal antibody that identifies sheep hematopoietic stem/progenitor cells.

OBJECTIVE: We and many others have long used sheep as a predictive model system in which to explore stem cell transplantation. Unfortunately, while numerous markers are available to identify and isolate human hematopoietic stem cells (HSC), no reagents exist that allow HSC/progenitors from sheep to be identified or purified, greatly impeding the application of this well-established large animal model to the study of autologous or allogeneic HSC transplantation. The current studies were undertaken to create a monoclonal antibody to sheep CD34 that would enable isolation and study of sheep HSC/progenitors. MATERIALS AND METHODS: A partial cDNA to the extracellular domain of the sheep CD34 antigen was polymerase chain reaction cloned, characterized, and used to genetically immunize mice and create hybridomas. RESULTS: The resultant monoclonal antibody to sheep CD34 allows flow cytometric detection of sheep HSC/progenitors present within bone marrow, cord blood, and mobilized peripheral blood. Moreover, this antibody can be used to enrich for HSC/progenitors with enhanced in vitro colony-forming potential, and also identifies endothelial cells in situ within paraffin-embedded tissue sections, similarly to antibodies to human CD34. CONCLUSIONS: The availability of this monoclonal antibody recognizing the stem cell antigen CD34 in sheep will greatly facilitate the study of autologous and allogeneic HSC transplantation using this clinically relevant large animal model.

Efficient generation of human hepatocytes by the intrahepatic delivery of clonal human mesenchymal stem cells in fetal sheep.

UNLABELLED: Alternative methods to whole liver transplantation require a suitable cell that can be expanded to obtain sufficient numbers required for successful transplantation while maintaining the ability to differentiate into hepatocytes. Mesenchymal stem cells (MSCs) possess several advantageous characteristics for cell-based therapy and have been shown to be able to differentiate into hepatocytes. Thus, we investigated whether the intrahepatic delivery of human MSCs is a safe and effective method for generating human hepatocytes and whether the route of administration influences the levels of donor-derived hepatocytes and their pattern of distribution throughout the parenchyma of the recipient's liver. Human clonally derived MSCs were transplanted by an intraperitoneal (n = 6) or intrahepatic (n = 6) route into preimmune fetal sheep. The animals were analyzed 56-70 days after transplantation by immunohistochemistry, enzyme-linked immunosorbent assay, and flow cytometry. The intrahepatic injection of human MSCs was safe and resulted in more efficient generation of hepatocytes (12.5% +/- 3.5% versus 2.6% +/- 0.4%). The animals that received an intrahepatic injection exhibited a widespread distribution of hepatocytes throughout the liver parenchyma, whereas an intraperitoneal injection resulted in a preferential periportal distribution of human hepatocytes that produced higher amounts of albumin. Furthermore, hepatocytes were generated from MSCs without the need to first migrate/lodge to the bone marrow and give rise to hematopoietic cells. CONCLUSION: Our studies provide evidence that MSCs are a valuable source of cells for liver repair and regeneration and that, by the alteration of the site of injection, the generation of hepatocytes occurs in different hepatic zones, suggesting that a combined transplantation approach may be necessary to successfully repopulate the liver with these cells.

The human-sheep chimeras as a model for human stem cell mobilization and evaluation of hematopoietic grafts' potential.

OBJECTIVE: To investigate whether the sheep xenograft model of human hematopoiesis can be used to mimic mobilization of human hematopoietic stem cells in vivo. MATERIAL AND METHODS: Sheep transplanted with 3.6 x 10(6) CD34+ from human adult bone marrow were mobilized 1.5 years posttransplantation with human granulocyte colony-stimulating factor for 5 days. At day 3 and 4 of mobilization, human cells were harvested from peripheral blood (PB) and bone marrow (BM) and were injected into secondary sheep recipients (n = 6) and these animals were analyzed for the presence of human cells in their BM and PB, starting at 3.5 months posttransplantation. RESULTS: Maximum mobilization of human cells in PB occurred at day 3, with a 21-fold increase in total numbers of human cells, and a recovery of 5.5 x 10(4)/mL CD34+. In the BM, maximal numbers of human cells were achieved at day 4, with a 6.3-fold increase and a recovery of 1.5 x 10(4)/mL CD34+ cells. PB and BM mobilized human cells were then transplanted into new sheep recipients, and analysis at 3.5 months posttransplantation demonstrated that levels of human cell engraftment in BM of the group transplanted with mobilized PB were significantly lower than those transplanted with BM cells (0.6% +/- 0.1% vs 8.0% +/- 1.8%). Furthermore, in sheep transplanted with mobilized PB, the levels of human cells in circulation remained 2.5-fold higher than the levels of human cells found in their BM. CONCLUSION: Mobilization of human cells in the sheep model parallels human PB and BM hematopoietic stem cells (HSC) mobilization in healthy human donors in their ability to engraft, differentiate, and repopulate secondary hosts. Thus, this model can become a useful tool to study mobilization regimens, mechanisms, and quality of products obtained.

A human bone marrow mesodermal-derived cell population with hemogenic potential.

The presence, within the human bone marrow, of cells with both endothelial and hemogenic potential has been controversial. Herein, we identify, within the human fetal bone marrow, prior to establishment of hematopoiesis, a unique APLNR+, Stro-1+ cell population, co-expressing markers of early mesodermal precursors and/or hemogenic endothelium. In adult marrow, cells expressing similar markers are also found, but at very low frequency. These adult-derived cells can be extensively culture expanded in vitro without loss of potential, they preserve a biased hemogenic transcriptional profile, and, upon in vitro induction with OCT4, assume a hematopoietic phenotype. In vivo, these cells, upon transplantation into a fetal microenvironment, contribute to the vasculature, and generate hematopoietic cells that provide multilineage repopulation upon serial transplantation. The identification of this human somatic cell population provides novel insights into human ontogenetic hematovascular potential, which could lead to a better understanding of, and new target therapies for, malignant and nonmalignant hematologic disorders.

Correction: A human bone marrow mesodermal-derived cell population with hemogenic potential.

At the time of publication the funding information was omitted from the article - this has now been corrected in both the HTML and the PDF.

The time course of engraftment of human mesenchymal stem cells in fetal heart demonstrates that Purkinje fiber aggregates derive from a single cell and not multi-cell homing.

OBJECTIVE: To study the early time course of engraftment of human mesenchymal stem cells in fetal sheep heart and determine the relative roles of proliferation and homing in formation of aggregates of human Purkinje fiber cells. METHODS: The human sheep xenograft model was utilized for these studies. Prior to injection in the preimmune fetus, human cells were labeled with fluorescent dyes to be able to track human cells at early times of engraftment. RESULTS: Human stem cells were detected in fetal hearts between 29 and 39 hours after intraperitoneal injection. Engraftment was primarily in the Purkinje fiber system. By 45 hours engrafted human cells had a cardiac phenotype. When two groups of human mesenchymal stem cells, each labeled with a different fluorescent dye, were combined prior to injection, aggregates of human Purkinje fiber cells contained cells labeled with either one dye or the other, no aggregate contained cells labeled with both dyes. CONCLUSIONS: Human mesenchymal stem cells introduced into fetal sheep rapidly enter the myocardium. The swift differentiation into a cardiac phenotype indicates that the cardiac milieu has a strong influence on the fate of engrafting human mesenchymal stem cells. The absence of any aggregates of human Purkinje fiber cells containing both fluorescent dyes demonstrates that each aggregate of human Purkinje fiber cells is derived from a single mesenchymal stem cell and not from homing of multiple cells to a hotspot.

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.

A human stromal-based serum-free culture system supports the ex vivo expansion/maintenance of bone marrow and cord blood hematopoietic stem/progenitor cells.

OBJECTIVE: We investigated the role of human stromal layers (hu-ST) on the ex vivo expansion/maintenance of human hematopoietic stem/progenitor cells (HSC) from adult bone marrow (BM) and umbilical cord blood (CB). MATERIALS AND METHODS: BM and CB CD34(+)-enriched cells were cultured in serum-free medium supplemented with SCF, bFGF, LIF, and Flt-3, in the presence or absence of stroma, and analyzed for proliferation, phenotype, and clonogenic potential. RESULTS: Significant expansion of BM and CB CD34(+) and CD34(+)CD38(-) cells were achieved in the presence of hu-ST. The differentiative potential of both BM and CB CD34(+)-enriched cells cocultured with hu-ST was primarily shifted toward the myeloid lineage, while maintaining/expanding a CD7(+) population. Clonogenic analysis of the expanded cells showed increases in progenitors of the myeloid lineage, including colony-forming unit-granulocyte, macrophage (CFU-GM) and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-Mix) for both BM (stroma and stroma-free conditions) and CB cells in the presence of stroma. CONCLUSIONS: These results indicate that adult hu-ST in the presence of appropriate cytokines can be used to efficiently expand/maintain myeloid and lymphoid cell populations from human BM and CB HSC.

Boosting Hematopoietic Engraftment after in Utero Transplantation through Vascular Niche Manipulation.

In utero hematopoietic stem/progenitor cell transplantation (IUHSCT) has only been fully successful in the treatment of congenital immunodeficiency diseases. Using sheep as a large animal model of IUHSCT, we demonstrate that administration of CD146(+)CXCL12(+)VEGFR2(+) or CD146(+)CXCL12(+)VEGFR2(-) cells prior to, or in combination with, hematopoietic stem/progenitor cells (HSC), results in robust CXCL12 production within the fetal marrow environment, and significantly increases the levels of hematopoietic engraftment. While in the fetal recipient, donor-derived HSC were found to reside within the trabecular bone, the increased expression of VEGFR2 in the microvasculature of CD146(+)CXCL12(+)VEGFR2(+) transplanted animals enhanced levels of donor-derived hematopoietic cells in circulation. These studies provide important insights into IUHSCT biology, and demonstrate the feasibility of enhancing HSC engraftment to levels that would likely be therapeutic in many candidate diseases for IUHSCT.

Human mesenchymal stem cells form Purkinje fibers in fetal sheep heart.

BACKGROUND: We have investigated the usefulness of a model of cardiac development in a large mammal, sheep, for studies of engraftment of human stem cells in the heart. METHODS AND RESULTS: Adult and fetal human mesenchymal stem cells were injected intraperitoneally into sheep fetuses in utero. Hearts at late fetal development were analyzed for engraftment of human cells. The majority of the engrafted cells of human origin formed segments of Purkinje fibers containing exclusively human cells. There were no differences in engraftment of human mesenchymal stem cells from adult bone marrow, fetal brain, and fetal liver. On average, 43.2% of the total Purkinje fibers in random areas (n=11) of both ventricles were of human origin. In contrast, approximately 0.01% of cardiomyocytes were of human origin. CONCLUSIONS: Human mesenchymal stem cells preferentially engraft at high levels in the ventricular conduction system during fetal development in sheep. These findings raise the possibility that stem cells contribute to normal development of the fetal heart.

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.