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

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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.

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.

Pediatric End-Stage Failing Hearts Demonstrate Increased Cardiac Stem Cells.

BACKGROUND: We sought to determine the location, expression, and characterization of cardiac stem cells (CSCs) in children with end-stage heart failure (ESHF). We hypothesized ESHF myocardium would contain an increased number of CSCs relative to age-matched healthy myocardium, and ESHF-derived CSCs would have diminished functional capacity as evidenced by reduced telomere length. METHODS: Tissue samples were obtained from the explanted hearts of children undergoing heart transplantation with ESHF, defined as New York Heart Association class III or IV and ejection fraction less than 0.20, and from age-matched congenital heart disease patients with normal myocardium. The expression profile of cardiac-specific stem cell markers was determined using quantitative real time polymerase chain reaction and immunofluorescence. Cardiac stem cell growth reserve was assessed with telomere length. RESULTS: There were 15 ESHF and 15 age-matched congenital heart disease patients. End-stage heart failure myocardium demonstrated increased expression of c-kit(+) and islet-1(+) CSCs by 2.0- and 2.5-fold, respectively, compared with myocardium from congenital heart disease patients. There was no difference in expression of c-kit(+) CSCs with advancing age from infants to children in ESHF myocardium. The c-kit(+) CSCs isolated from ESHF patients demonstrated significantly reduced telomere length, suggesting a diminished functional capability in these cells (8.1 ± 0.6 kbp versus 6.3 ± 0.3 kbp; p = 0.015). CONCLUSIONS: End-stage heart failure myocardium demonstrated an age-independent increase in CSCs relative to healthy myocardium; however, these CSCs from ESHF patients may have diminished proliferative ability and reduced functionality as an autologous cell therapy candidate. Further investigation is necessary to determine the role of ESHF-derived CSCs within the myocardium.

Cardiosphere-derived cells from pediatric end-stage heart failure patients have enhanced functional activity due to the heat shock response regulating the secretome.

We have demonstrated that human neonatal cardiosphere-derived cells (CDCs) derived from the young are more regenerative due to their robust secretome. However, it is unclear how the decompensated pediatric heart impacts the functional activity of their CDCs. Our aim was to characterize the potency of pediatric CDCs derived from normal functioning myocardium of control heart disease (CHD) patients to those generated from age-matched end stage heart failure (ESHF) patients and to determine the mechanisms involved. ESHF-derived CDCs contained a higher number of c-kit(+) , Islet-1(+) , and Sca-1(+) cells. When transplanted into an infarcted rodent model, ESHF-derived CDCs significantly demonstrated higher restoration of ventricular function, prevented adverse remodeling, and enhanced angiogenesis when compared with CHD patients. The superior functional recovery of the ESHF-derived CDCs was mediated in part by increased SDF-1α and VEGF-A secretion resulting in augmented recruitment of endogenous stem cells and proliferation of cardiomyocytes. We determined the mechanism is due to the secretome directed by the heat shock response (HSR), which is supported by three lines of evidence. First, gain of function studies demonstrated that increased HSR induced the lower functioning CHD-derived CDCs to significantly restore myocardial function. Second, loss-of function studies targeting the HSR impaired the ability of the ESHF-derived CDCs to functionally recover the injured myocardium. Finally, the native ESHF myocardium had an increased number of c-kit(+) cardiac stem cells. These findings suggest that the HSR enhances the functional activity of ESHF-derived CDCs by increasing their secretome activity, notably SDF-1α and VEGF-A.

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.

Mesenchymal stem cells engineered to inhibit complement-mediated damage.

Mesenchymal stem cells (MSC) preferentially migrate to damaged tissues and, due to their immunomodulatory and trophic properties, contribute to tissue repair. Although MSC express molecules, such as membrane cofactor protein (CD46), complement decay-accelerating factor (CD55), and protectin (CD59), which confer protection from complement-mediated lysis, MSC are recruited and activated by anaphylatoxins after transplantation, potentially causing MSC death and limiting therapeutic benefit. We have previously demonstrated that transduction of MSC with a retrovirus encoding HCMV-US proteins resulted in higher levels of MSC engraftment due to decreased HLA-I expression. Here, we investigate whether engineering MSC to express US2 (MSC-US2), US3 (MSC-US3), US6 (MSC-US6), or US11 (MSC-US11) HCMV proteins can alter complement recognition, thereby better protecting MSC from complement attack and lysis. HCMV-US proteins increased MSC CD59 expression at different levels as determined by flow cytometric evaluation of the median fluorescence intensity ratio (MFI). A significant increase in CD59 expression was seen in MSC-US2, MSC-US3, and MSC-US6, but not in MSC-US11. Only MSC-US2 displayed increased expression of CD46, while US2 and US3 proteins were both able to augment the percentage of MSC expressing this molecule. Regardless of the HCMV protein expressed, none changed CD55 MFI; however, expression of US6, US11, and US2 each increased the percentage of MSC that were positive for this molecule. Because US2 protein was the most efficient in up-regulating all three complement regulatory proteins, we used a functional complement-mediated cytotoxicity assay to investigate whether MSC-US2 were protected from complement-mediated lysis. We demonstrated that over-expression of the US2 protein reduced complement lysis by 59.10±12.89% when compared to untransduced MSC. This is the first report, to our knowledge, describing a role of HCMV-US proteins in complement evasion, and our data shows that over-expression of US2 protein on MSC could serve as a strategy to protect these cells from complement lysis.

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.

Modulation of human mesenchymal stem cell immunogenicity through forced expression of human cytomegalovirus us proteins.

BACKGROUND: Mesenchymal stem cells (MSC) are promising candidates for cell therapy, as they migrate to areas of injury, differentiate into a broad range of specialized cells, and have immunomodulatory properties. However, MSC are not invisible to the recipient's immune system, and upon in vivo administration, allogeneic MSC are able to trigger immune responses, resulting in rejection of the transplanted cells, precluding their full therapeutic potential. Human cytomegalovirus (HCMV) has developed several strategies to evade cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell recognition. Our goal is to exploit HCMV immunological evasion strategies to reduce MSC immunogenicity. METHODOLOGY/PRINCIPAL FINDINGS: We genetically engineered human MSC to express HCMV proteins known to downregulate HLA-I expression, and investigated whether modified MSC were protected from CTL and NK attack. Flow cytometric analysis showed that amongst the US proteins tested, US6 and US11 efficiently reduced MSC HLA-I expression, and mixed lymphocyte reaction demonstrated a corresponding decrease in human and sheep mononuclear cell proliferation. NK killing assays showed that the decrease in HLA-I expression did not result in increased NK cytotoxicity, and that at certain NK∶MSC ratios, US11 conferred protection from NK cytotoxic effects. Transplantation of MSC-US6 or MSC-US11 into pre-immune fetal sheep resulted in increased liver engraftment when compared to control MSC, as demonstrated by qPCR and immunofluorescence analyses. CONCLUSIONS AND SIGNIFICANCE: These data demonstrate that engineering MSC to express US6 and US11 can be used as a means of decreasing recognition of MSC by the immune system, allowing higher levels of engraftment in an allogeneic transplantation setting. Since one of the major factors responsible for the failure of allogeneic-donor MSC to engraft is the mismatch of HLA-I molecules between the donor and the recipient, MSC-US6 and MSC-US11 could constitute an off-the-shelf product to overcome donor-recipient HLA-I mismatch.

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.

Expression levels of the PiT-2 receptor explain, in part, the gestational age-dependent alterations in transduction efficiency after in utero retroviral-mediated gene transfer.

BACKGROUND: A fundamental obstacle to using retroviral-mediated gene transfer (GT) to treat human diseases is the relatively low transduction levels that have been achieved in clinically relevant human cells. We previously showed that performing GT in utero overcomes this obstacle and results in significant levels of transduction within multiple fetal organs, with different tissues exhibiting optimal transduction at different developmental stages. We undertook the present study aiming to elucidate the mechanism for this age-dependent transduction, testing the two factors that we hypothesized could be responsible: (i) the proliferative status of the tissue at the time of GT and (ii) the expression level of the amphotropic PiT-2 receptor. METHODS: Immunofluorescence was performed on tissues from sheep of varying developmental stages to assess the proliferative status of the predominant cells within each organ as a function of age. After developing an enzyme-linked immunosorbent assay (ELISA) and a quantitative reverse transcription chain reaction (qRT-PCR) assay, we then quantified PiT-2 expression at the protein and mRNA levels, respectively. RESULTS: The results obtained indicate that the proliferative status of organs at the time of fetal GT is not the major determinant governing transduction efficiency. By contrast, our ELISA and qRT-PCR analyses demonstrated that PiT-2 mRNA and protein levels vary with gestational age, correlating with the observed differences in transduction efficiency. CONCLUSIONS: The findings of the present study explain the age-related differences that we previously observed in transduction efficiency after in utero GT. They also suggest it may be possible to achieve relatively selective GT to specific tissues by performing in utero GT when levels of PiT-2 are maximal in the desired target organ.

Phenotypic correction of hemophilia A in sheep by postnatal intraperitoneal transplantation of FVIII-expressing MSC.

We recently re-established a line of sheep that accurately mimics the clinical symptoms and genetics of severe hemophilia A (HA). Here, we tested a novel, nonablative transplantation therapy in two pediatric HA animals. Paternal mesenchymal stem cells (MSC) were transduced with a porcine FVIII-encoding lentivector and transplanted via the intraperitoneal route without preconditioning. At the time of transplantation, these animals had received multiple human FVIII treatments for various spontaneous bleeds and had developed debilitating hemarthroses, which produced severe defects in posture and gait. Transplantation of transduced MSC resolved all existent hemarthroses, and spontaneous bleeds ceased. Damaged joints recovered fully; the animals regained normal posture and gait and resumed normal activity. Despite achieving factor-independence, a sharp rise in pre-existent Bethesda titers occurred following transplantation, decreasing the effectiveness and duration of therapy. Postmortem examination revealed widespread engraftment, with MSC present within the lung, liver, intestine, and thymus, but particularly within joints affected at the time of transplantation, suggesting MSC homed to sites of ongoing injury/inflammation to release FVIII, explaining the dramatic improvement in hemarthrotic joints. In summary, this novel, nonablative MSC transplantation was straightforward, safe, and converted life-threatening, debilitating HA to a moderate phenotype in a large animal model.

Characterization and functionality of cardiac progenitor cells in congenital heart patients.

BACKGROUND: Human cardiac progenitor cells (hCPCs) may promote myocardial regeneration in adult ischemic myocardium. The regenerative capacity of hCPCs in young patients with nonischemic congenital heart defects for potential use in congenital heart defect repair warrants exploration. METHODS AND RESULTS: Human right atrial specimens were obtained during routine congenital cardiac surgery across 3 groups: neonates (age, <30 days), infants (age, 1 month to 2 years), and children (age, >2 to ≤13 years). C-kit(+) hCPCs were 3-fold higher in neonates than in children >2 years of age. hCPC proliferation was greatest during the neonatal period as evidenced by c-kit(+) Ki67(+) expression but decreased with age. hCPC differentiation capacity was also greatest in neonatal right atrium as evidenced by c-kit(+), NKX2-5(+), NOTCH1(+), and NUMB(+) expression. Despite the age-dependent decline in resident hCPCs, we isolated and expanded right atrium-derived CPCs from all patients (n=103) across all ages and diagnoses using the cardiosphere method. Intact cardiospheres contained a mix of heart-derived cell subpopulations that included cardiac progenitor cells expressing c-kit(+), Islet-1, and supporting cells. The number of c-kit(+)-expressing cells was highest in human cardiosphere-derived cells (hCDCs) grown from neonatal and infant right atrium. Furthermore, hCDCs could differentiate into diverse cardiovascular lineages by in vitro differentiation assays. Transplanted hCDCs promoted greater myocardial regeneration and functional improvement in infarcted myocardium than transplanted cardiac fibroblasts. CONCLUSIONS: Resident hCPCs are most abundant in the neonatal period and rapidly decrease over time. hCDCs can be reproducibly isolated and expanded from young human myocardial samples regardless of age or diagnosis. hCPCs are functional and have potential in congenital cardiac repair.

Generation of tissue-specific cells from MSC does not require fusion or donor-to-host mitochondrial/membrane transfer.

Human mesenchymal stem cells (MSC) hold great promise for cellular replacement therapies. Despite their contributing to phenotypically distinct cells in multiple tissues, controversy remains regarding whether the phenotype switch results from a true differentiation process. Here, we studied the events occurring during the first 120 h after human MSC transplantation into a large animal model. We demonstrate that MSC, shortly after engrafting different tissues, undergo proliferation and rapidly initiate the differentiative process, changing their phenotype into tissue-specific cells. Thus, the final level of tissue-specific cell contribution is not determined solely by the initial level of engraftment of the MSC within that organ, but rather by the proliferative capability of the ensuing tissue-specific cells into which the MSC rapidly differentiate. Furthermore, we show that true differentiation, and not cell fusion or transfer of mitochondria or membrane-derived vesicles between transplanted and resident cells, is the primary mechanism contributing to the change of phenotype of MSC upon transplantation.

Factors determining the risk of inadvertent retroviral transduction of male germ cells after in utero gene transfer in sheep.

The possibility of permanent genetic changes to the germline is central to the bioethics of in utero gene therapy (IUGT) because of the concern of inadvertent potentially deleterious alterations to the gene pool. Despite presumed protection of the male germline due to early germ cell (GC) compartmentalization, we reported that GCs within the developing ovine testes are transduced at low levels after retrovirus-mediated IUGT, thus underscoring the need for a thorough understanding of GC development in clinically predictive models to determine the optimal time to perform IUGT and avoid germline modification. In the present studies, we used the fetal sheep model to analyze GCs for phenotype, location, proliferation, and incidence of transduction after IUGT at various fetal ages to learn when during development the nascent germline is likely to be at greatest risk of retrovirus-mediated alteration. Our studies show that although GCs were transduced at all injection ages, the levels of transduction varied by nearly 700-fold as a function of the age at transfer. After remaining largely quiescent as they migrated to/settled within nascent sex cords, GCs began active cycling before cord closure was complete, suggesting this is likely the point at which they would be most susceptible to retroviral transduction.Furthermore, we observed that compartmentalization of GCs continued into early postnatal life, suggesting the male germline may be vulnerable to low-level inadvertent retroviral vector modification throughout fetal life, but that this risk can be minimized by performing IUGT later in gestation.

Early fetal gene delivery utilizes both central and peripheral mechanisms of tolerance induction.

OBJECTIVE: We previously reported the induction of stable immune tolerance following direct injection of retroviral vectors into preimmune fetal sheep. In the present studies, we conduct detailed analysis of the thymus of recipients of in utero gene transfer (IUGT) to delineate the mechanism of the observed immune tolerance and assess the impact of recipient age on this process. MATERIALS AND METHODS: Fetal sheep at varying gestational ages received the MSCV-NeoR-RFP retroviral vector. The thymus was then collected from these animals at 27 to 30 days postinjection and analyzed for evidence of transduction of key immunoregulatory thymic cells. RESULTS: Our results reveal that both thymic epithelial cells (TEC), crucial for presentation of self-antigen during T-cell thymic selection, and the cells comprising the Hassall's corpuscles, which can present antigen directly and also instruct dendritic cells to induce the formation of CD4(+)CD25(+) T-regulatory cells in the thymus, were only efficiently transduced if IUGT was performed early in gestation. CONCLUSIONS: Our findings thus demonstrate, for the first time, that early IUGT can potentially take advantage of multiple tolerogenic avenues in the fetus, transducing both TEC, which promote central tolerance, and Hassall's corpuscles, which induce formation of T regulatory cells that could act to maintain peripheral tolerance to the transgene products.

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 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 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.