Preimmune Fetal Sheep Research Articles

Generation of CD34+ cells from human embryonic stem cells using a clinically applicable methodology and engraftment in the fetal sheep model

Until now, exvivo generation of CD34(+) hematopoietic stem cells (HSCs) from human embryonic stem cells (hESCs) mostly involved use of feeder cells of nonhuman origin. Although they provided invaluable models to study hematopoiesis, invivo engraftment of hESC-derived HSCs remains a challenging task. In this study, we used a novel coculture system composed of human bone marrow-derived mesenchymal stromal/stem cells (MSCs) and peripheral blood CD14(+) monocyte-derived macrophages to generate CD34(+) cells from hESCs invitro. Human ESC-derived CD34(+) cells generated using this method expressed surface makers associated with adult human HSCs and upregulated hematopoietic stem cell genes comparable to human bone marrow-derived CD34(+) cells. Finally, transplantation of purified hESC-derived CD34(+) cells into the preimmune fetal sheep, primed with transplantation of MSCs derived from the same hESC line, demonstrated multilineage hematopoietic activity with graft presence up to 16 weeks after transplantation. This invivo demonstration of engraftment and robust multilineage hematopoietic activity by hESC-derived CD34(+) cells lends credence to the translational value and potential clinical utility of this novel differentiation and transplantation protocol.

Intramyocardial Transplantation and Tracking of Human Mesenchymal Stem Cells in a Novel Intra-Uterine Pre-Immune Fetal Sheep Myocardial Infarction Model: A Proof of Concept Study

Although stem-cell therapies have been suggested for cardiac-regeneration after myocardial-infarction (MI), key-questions regarding the in-vivo cell-fate remain unknown. While most available animal-models require immunosuppressive-therapy when applying human cells, the fetal-sheep being pre-immune until day 75 of gestation has been proposed for the in-vivo tracking of human cells after intra-peritoneal transplantation. We introduce a novel intra-uterine myocardial-infarction model to track human mesenchymal stem cells after direct intra-myocardial transplantation into the pre-immune fetal-sheep. Thirteen fetal-sheep (gestation age: 70–75 days) were included. Ten animals either received an intra-uterine induction of MI only (n = 4) or MI+intra-myocardial injection (IMI;n = 6) using micron-sized, iron-oxide (MPIO) labeled human mesenchymal stem cells either derived from the adipose-tissue (ATMSCs;n = 3) or the bone-marrow (BMMSCs;n = 3). Three animals received an intra-peritoneal injection (IPI;n = 3; ATMSCs;n = 2/BMMSCs;n = 1). All procedures were performed successfully and follow-up was 7–9 days. To assess human cell-fate, multimodal cell-tracking was performed via MRI and/or Micro-CT, Flow-Cytometry, PCR and immunohistochemistry. After IMI, MRI displayed an estimated amount of 1×105–5×105 human cells within ventricular-wall corresponding to the injection-sites which was further confirmed on Micro-CT. PCR and IHC verified intra-myocardial presence via detection of human-specific β-2-microglobulin, MHC-1, ALU-Sequence and anti-FITC targeting the fluorochrome-labeled part of the MPIOs. The cells appeared viable, integrated and were found in clusters or in the interstitial-spaces. Flow-Cytometry confirmed intra-myocardial presence, and showed further distribution within the spleen, lungs, kidneys and brain. Following IPI, MRI indicated the cells within the intra-peritoneal-cavity involving the liver and kidneys. Flow-Cytometry detected the cells within spleen, lungs, kidneys, thymus, bone-marrow and intra-peritoneal lavage, but not within the heart. For the first time we demonstrate the feasibility of intra-uterine, intra-myocardial stem-cell transplantation into the pre-immune fetal-sheep after MI. Utilizing cell-tracking strategies comprising advanced imaging-technologies and in-vitro tracking-tools, this novel model may serve as a unique platform to assess human cell-fate after intra-myocardial transplantation without the necessity of immunosuppressive-therapy.

Modulation of Human Mesenchymal Stem Cell Immunogenicity through Forced Expression of Human Cytomegalovirus US Proteins

BackgroundMesenchymal 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 FindingsWe 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 SignificanceThese 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.

Intravascular Route Is Not Superior to an Intraperitoneal Route for In Utero Transplantation of Human Hematopoietic Stem Cells and Engraftment in Sheep

Previously, we have reported in utero hematopoietic stem-cell transplantation (IUT) in sheep (1) and nonhuman primates (2) using an intraperitoneal (IP) route, as performed in clinical trials in humans (3,4) to treat congenital disorders, including X-linked severe combined immunodeficiency. Recent studies (5,6) have demonstrated that, for IUT in mice, the intravascular (IV) route facilitates the injection of cell doses higher than those that can be injected IP, thereby resulting in increased levels of donor-derived chimerism after birth. The studies are tremendous in that they have demonstrated the feasibility of such narrow route injection in mouse fetuses and yielded superior outcomes. However, no study has compared the IV and IP approaches with respect to the efficiency of engraftment in large animals. In this study, we compared the efficacy of these approaches for IUT in sheep. Human cord blood CD34+ cells were transplanted into the right ventricular cavity (IV group, n=4) or abdominal cavity (IP group, n=3) of the preimmune fetal sheep (at gestation days 59–61; full term, 147 days). The cells were injected through the uterine wall using a 23-gauge needle under ultrasound guidance. For the IV approach, we transplanted cells into the right ventricular cavity instead of the vitelline vein (5,6), because the vitelline vein was too small to be detected under ultrasound guidance at the gestation days. The number of cells transplanted per fetus ranged from 1.4×105 to 6.3×105; there were no intergroup differences. After birth (4 months after transplant), few transplant-derived cells were detected in the peripheral blood, but the engraftment could be properly examined by clonogenic hematopoietic progenitor assay to detect human hematopoietic colony-forming units in the bone marrow (BM). The fractions of human colony-forming units were 1.3%±0.1% and 1.3%±0.9% in the IP and IV groups, respectively (Table 1), indicating that there was no difference in the engraftment between the two groups.TABLE 1: In utero transplantation and engraftment of human hematopoietic stem cells in sheepInitially, we had hypothesized that the engraftment of the transplanted cells that were injected by the IV route in the BM at approximately day 60 of the gestation period in sheep would be better than that of the cells injected by the IP route because during this period, hematopoietic stem cells IV move from the liver into the BM (7). However, we found no difference in the efficacies of both approaches. Furthermore, bradycardia was often detected in the fetuses that were injected by the IV route, thereby implying that the IV approach might have some risk associated with it. Considering that both approaches are equally efficacious for the engraftment of the transplanted cells and because IP injection is evidently easier and safer than IV injection, we hypothesize that the IP approach is more meritorious than the IV approach for IUT experiments in large animals and clinical studies in humans. Therefore, we believe that further clinical trials of IUT can be conducted using the IP approach. Yujiro Tanaka1 Shigeo Masuda1 Tomoyuki Abe1,2,3 Satoshi Hayashi4 Yoshihiro Kitano4 Yoshikazu Nagao2,3 Yutaka Hanazono1 1 Division of Regenerative Medicine Center for Molecular Medicine Jichi Medical University Tochigi, Japan 2 Department of Agriculture Utsunomiya University Tochigi, Japan 3 Division of Biological Production Science United Graduate School of Agricultural Science Tokyo University of Agriculture and Technology Tokyo, Japan 4 Department of Obstetrics and Gynecology National Center for Child Health and Development Tokyo, Japan

EVALUATION OF ENGRAFTMENT AND DIFFERENTIATION OF DIFFERENT SOURCES OF HUMAN STEM CELLS IN FETAL SHEEP BRAIN

It is known that the brain can be affected by many diseases/disorders for which therapies are not yet available. Recently, different investigators have reported on the ability of stem cells to differentiate into different neural cell types in vitro and in vivo. This knowledge opened new perspectives in stem cell therapy for neurological disorders/diseases. Hematopoietic (HSC), mesenchymal (MSC) and neural (NSC) stem cells are the most promising source of stem cells given either their easiness of harvest or their differentiative ability. Upon grafting into the brain, stem cells could give rise directly to new neurons or could generate other cells of the central nervous system, such oligodendrocytes, or glial cells, that may contribute to neural growth. Furthermore these cells could be therapeutic by secreting missing substances or enzymes that are needed for the normal function of the brain. Since mesenchymal stem cells (MSCs) have been shown to suppress T-cell activation in vitro and in vivo, transplantation of these cells into the brain could led not only to a source of new neural cells but also could provide an anti-inflammatory response. In order to investigate the therapeutic potential of different sources of stem cells, pre-immune fetal sheep were transplanted by intraperitoneal injection, at 60 days of gestation, with HSC, NSC or MSC. Animals were euthanized at 60-80 days post transplantation and tissues were collected from different regions of the brain and analyzed by flow cytometry and immunohistochemistry using different antibodies for known neural markers. Levels of donor cell engraftment and types of neural cells obtained were then correlated to the source of the transplanted cells. We hope that these studies will be able to discern what is/are the best source(s) of stem cells to use for neural cell replacement therapies.

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

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. 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. 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. 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.

Gene Transfer Early in Gestation Results in Transduction of Key Cellular Mediators of Both Central and Peripheral Immune Tolerance.

We have previously reported that the direct intraperitoneal (IP) injection of murine stem cell virus (MSCV)-based retroviral vectors into preimmune fetal sheep results in the induction of stable long-term immune tolerance to an antigenic transgene product that enables post-natal antigen boosting without eliciting an immune response. These results suggested that in utero gene therapy (IUGT) may be an ideal approach for treating diseases such as the hemophilias, in which post-natal attempts at both gene therapy and protein replacement therapy have often been thwarted by the formation of immune inhibitors. In the present studies, we performed experiments to delineate the mechanism of the observed immune tolerance following IUGT and to define the optimal window during gestation when gene transfer should be performed to ensure induction of transgene immune tolerance. To this end, we performed IUGT on fetal sheep at various gestational ages (55–110 days of gestation; term: 150 days). At 30 days post-IUGT, sheep were euthanized and thymic cryosections analyzed by immunohistochemistry using an antibody specific to the vector-encoded NeoR transgene product (NPT) and either a pan-cytokeratin antibody to label thymic epithelial cells, or an anti-CD45 antibody to label thymocytes. We determined that thymic tissue is in fact transduced in the majority of animals following IUGT regardless of the age at which IUGT is performed. Importantly, however, efficient transduction of thymic epithelial cells that are thought to be one of the key cell types responsible for presentation of self-antigen during T cell thymic selection only occurred if the gene transfer was performed prior to 72 days of gestation (term: 150 days). If IUGT was performed at day 72 or later, transduction was limited to predominantly thymocytes. These results suggest that IUGT may only result in successful induction of central immune tolerance if performed prior to 72 days of gestation in this model. These ongoing studies have also revealed that epithelial-like thymic stromal lymphopoietin (TSLP)+ cells comprising the Hassall's corpuscles are also transduced if IUGT is performed early in gestation (prior to 72 days of gestation), but not if it is performed after that point. Since recent studies have shown that one of the main functions of the Hassall's corpuscles is to instruct dendritic cells to induce the formation of CD4+CD25+ T-regulatory cells in the thymus, we performed FACS analysis on the peripheral blood of animals that received IUGT at varying gestational ages, focusing on subtypes of immune effector cells. These analyses revealed that animals that received IUGT early in gestation (prior to 72 days) have significantly higher percentages of CD4+CD25+ cells within their periphery than do animals transduced later in gestation. This finding is particularly interesting, given that CD4+CD25+ cells are thought to be T-regulatory cells that are involved in maintenance of peripheral tolerance. In conclusion, we have shown that by performing IUGT early in gestation, one can take advantage of multiple tolerogenic avenues present in the fetus, transducing both thymic epithelial cells which may promote induction of central immune tolerance and cells of Hassall's corpuscles, which can then in turn lead to the instruction of dendritic cells to induce the formation of T-regulatory cells that can help maintain peripheral immune tolerance to the transgene products.

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

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. 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.

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

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). 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. 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. 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.

Proteomic Analysis Reveals Intrinsic Differences between Phenotypically Identical Mesenchymal Stem Cells.

We have previously reported the isolation of Stro-1+, CD45-, GlyA-human MSC from human bone marrow (BM), liver (LV), and brain (BR). Despite their similar morphology and phenotype, these cells, upon in vivo transplantation into the pre-immune fetal sheep model, seemed to differentiate more efficiently into cells of their tissue of origin. For instance, all 3 sources of MSC gave rise to blood, with average levels of human CD45+ cells in chimeric animals of 8.7% for LVMSC, 3.3% for BRMSC, and 4.5%for BMMSC. Human neural cells were also detected in the brain of transplanted animals with the highest levels obtained with BRMSC (1.33%) and lowest with LVMSC (0.4%). However, BRMSC generated very few human hepatocytes (2–10 hepatocytes/section), while both LVMSC and BMMSC gave rise to much higher levels of hepatocytes. This led us to hypothesize that despite the similar phenotype, the proteome makeup of MSC from different tissues may be distinct. To test this hypothesis, we examined the commonalities and differences in the protein repertoire of cultured MSC from these 3 different sources. The eighty most prominent protein spots from the 2-D gel of each cell population were removed and identified by MOLDI-TOF/TOF mass spectroscopy after Trypsin digestion. Our results demonstrate that there are a large number of common proteins between the LV, BM and BR MSC (> 75%). Besides the cytoskeletal and associative proteins such as Vimentin, β-actin, cofilin, Tropomyosin and Lamin A/C, the cells contained a large quantity of proteins for protein processing such as Cyclophilin A, Endoplasmic reticulum protein ERp29, Disulfide-isomerase ER60 and Glucose-regulated proteins 75 and 78, which may be important for the broad differentiative potential of MSC. Despite these similarities, forty percent of the identified proteins showed significantly different expression levels between the three types of cells (> 300%), the majority of which were not proteins previously shown to be BM-, Liver or Brain-specific. Interestingly, however, LV-derived cells contained a 5-fold greater concentration of Albumin than BM or Brain cells, which may explain their greater tendency to form liver tissue in vivo. Likewise, since BMMSC were enriched in smooth muscle associated proteins, potentially they may constitute a better source to be used for muscle repair. Thus, the definition of the different MSC proteomes can lead not only to the discover of critical differences involved in differentiation/plasticit into different cell fates, but may also help us to identify distinct cell populations that are ideally suited for cell therapies for specific organs.

750. Expression Levels of the Amphotropic Receptor May Explain Gestational Age-Dependence of Tissue Transduction in Fetal Sheep

We have recently reported that the gestational age of the recipient determines the pattern and level of transgene expression following in utero retroviral gene transfer by direct injection into pre-immune fetal sheep (Molecular Therapy 11(2):284-293, 2005). These studies suggest that the developmental stage of each organ at the time of injection may determine its susceptibility to in utero gene transfer, and that each organ possesses its own unique temporal window during which optimal transduction can be achieved. In the present studies, we sought to determine the mechanism by which the recipient's age was affecting transduction efficiency and subsequent transgene expression. One can envision two mechanisms whereby the age of the tissue could affect its susceptibility to transduction with murine retroviral vectors. The first of these is the proliferative state of the tissue, since these vectors require cell division and nuclear membrane breakdown for genomic integration. To investigate this possibility, we have begun performing analysis of the fetal liver and lung at various gestational ages using BrdU labeling and staining of tissue sections with antibodies to Ki-67 and PH3, both markers of mitosis. Thus far, our results provide evidence that the proliferative state of the various tissues is not the primary mechanism by which transduction efficiency is being limited, since no significant difference was observed in the percentage of cells labeled in each tissue at the various ages studied. The other primary mechanism whereby the age of the tissue could alter its susceptibility to transduction is through age-dependent alterations in the levels of the receptor used for viral attachment and entry, namely, the amphotropic receptor, PiT2. To test this hypothesis, we custom synthesized an antibody to the ovine PiT2 receptor and developed an ELISA to evaluate the various fetal tissues at different gestational ages to determine whether the age of each tissue impacts upon its levels of PiT2 expression. Thus far, our results suggest that peaks in the levels of PiT2 expression occur at different stages of development in different tissues, thereby providing a possible explanation for the observed alterations in transduction efficiency. We are currently conducting real-time RT-PCR assays to confirm these alterations in the levels of PiT2 expression as a function of gestational age. It is hoped that these ongoing studies will enable us to determine the optimal stages for gene delivery to each of the major organs of the developing fetus and delineate the mechanisms that govern the levels of gene transfer that can be obtained.

Generation of Robust Numbers of Functional Human Hepatocytes by Human Hematopoietic Stem Cells in the Non-Injury Fetal Sheep Model.

We have previously shown that human hematopoietic stem cell preparations (HSCs) are able to engraft and transdifferentiate into human hepatocyte in the human/sheep xenograft model (Blood. 2004 Jul 1 Epub). In the present study 1x106 human CD34+ cells were transduced with GFP expressing RD114 pseudotyped spleen fociforming retroviral vector in vitro and injected into pre-immune fetal sheep. Two months post-transplantation, immunohistochemistry confirmed production of human hepatocytes that were double positive for GFP and Hepar1. These data indicate that transduced human CD34+ cells are able to transdifferentiate into human hepatocyte and still be able to express GFP. In order to retrieve human hepatocytes from the liver of chimeric sheep, we used c-met antibody to sort out human hepatocytes. Flowcytometric analysis demonstrated 8% c-met+ cells in the liver of chimeric sheep. Preliminary data using PCR analysis from c-met+ cells for human beta-2 microglobulin demonstrated presence of human cells in c-met+ population. Livers from chimeric sheep were treated with collagenase to obtain single cell suspension and 3x105 cells/ml were placed in T75 flask covered with 0.2% gelatin and cultured in optimized media designed to support the proliferation and maintenance of human and sheep hepatocytes. In this system, using a weekly passage protocol, we were able to maintain/expand human (and sheep) hepatocytes for up to 3 months. The cells are able to produce human albumin in culture and are CK19 positive, a-fetoprotein negative, and cytochrome P450 negative. These results demonstrate that in this non-injury large animal model human HSCs generate robust numbers of functional human hepatocytes apparently via a “transdifferentiation” mechanism.

Pluripotent Unrestricted Somatic Stem Cells (USSC) from Cord Blood Generate Hepatocytes in Vivo, but In Vitro Convert into Immature Endodermal Precursors: The Critical Role of the Biological Niche.

As shown recently by our group (Koegler et al. 2004; J. Exp. Med. 200: 19:1–13) transplantation of USSC in a non-injury model, the preimmune fetal sheep, resulted in more than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion. Here we examined whether we are able to trigger USSC in vitro into the an endodermal differentiation pathway applying protocols described for both embryonal as well as adult stem cell. The following matrix/growth factors/organic substances were applied: Neurobasal medium/supplement B27/bFGF with and without nicotinamid, on fibronectin, laminin or matrigel for differentiation into the pancreatic development as well as HGF, NGF, bFGF and HGF, FGF4 with or without Oncostatin M to induce liver development. 15 different USSC lines were differentiated for 1, 2, 3, 4 and 6 weeks. Primers were designed with the strategy to define stages of endodermal development on the basis of the embryonic cell development from mouse and human. Therefore the following markers were established: GATA 4, HNF-1, HNF3ß and HNF 4a to define the embryonic and visceral (extra-embryonic) endoderm, a common precursor phenotype for both liver/exocrine pancreas development. To further assess differentiation into liver cells, a-1 antitrypsin, a-fetoprotein, albumin, HGF, Cyp2B6, Cyp3A4, Gys2 and PDX-1, PAX4, ISL-1, NKx6.1, NeuroD, insulin to determine differentiation into the pancreatic development as well as epithelial markers as CK8, CK18, CK19 were analyzed by RT-PCR and subsequent hybridization using gene-specific oligonucleotide probes. USSC were tested negativ for the majority of the markers, only HGF and cytokeratin markers CK8/18 and CK19 were tested positiv. In vitro differentiation shows that USSC never expressed Neuro D, HNF1, HNF3b, PDX-1, PAX4, insulin and a-fetoprotein, but do express depending on the culture conditions common endodermal precursor markers HNF4a, GATA4 (but not HNF1 or HNF3ß) as well as albumin, Cyp2B6, Cyp3A4, Gys2 (liver development) and Nkx6.1 and ISL-1 (pancreatic development. It is very interesting to note that ISL-1, required for the formation of the dorsal mesenchym and essential for the dorsal exocrine pancreas development is strong expressed, although all other markers as PDX-1 and PAX4 were always tested negative. We have never observed the expression of a-fetoprotein, which might be explained by the kinetic of this factor (Young-Yang, Nat.Cell Biology 2004). In summary, the results show that under the vitro conditions applied, only an endodermal precursor phenotype could be established. Therefore at present co-cultivation experiments with injured hepatocytes from sheep/rat/human are performed to mimic the biological niche in vitro as shown by Young-Yang et al. Western blots with an human albumin specific antibody (moAb, clone HAS-11, Sigma) revealed that this approach resulted in an strong expression of albumin in USSC. Moreover, this co-cultivation model is useful for the identification and characterization of factors who are responsible for the biological niche in vivo.

Human Embryonic Stem Cell (hESC)-Derived Hematopoietic Elements Are Capable of Engrafting Primary as well as Secondary Fetal Sheep Recipients.

We used transplantation into 10 and 20 pre-immune fetal sheep recipients (55–65 days-old, term: 145 days) to evaluate the in vivo potential of hematopoietic elements derived from hESC. The in utero human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and post-natal human sources. Five transplant groups were established. Non-differentiated hESC were injected in one group. In the second and third group, embroid bodies differentiated for 8 days were injected whole or CD34+ cells were selected for injection. In the fourth and fifth group, hESC were differentiated on S17 mouse stroma layer and injected whole or CD34+ cells were selected for injection. The animals were allowed to complete gestation and be born. Bone marrow and peripheral blood samples were taken periodically up to over 12 months after injection, and PCR and flowcytometry was used to determine the presence of human DNA/blood cells in these samples. A total of 30 animals were analyzed. One primary recipient that was positive for human hematopoietic activity was sacrificed and whole bone marrow cells were transplanted into a secondary recipient. We analyzed the secondary recipient at 9 months post-injection by PCR and found it to be positive for human DNA in its peripheral blood and bone marrow. This animal was further challenged with human GM-CSF and human hematopoietic activity was noted by flowcytometry analyses of bone marrow and peripheral blood samples. Further, CD34+ cells enriched from its bone marrow were cultured in methylcellulose and human colonies were identified by PCR. We therefore conclude that hESC are capable of generating hematopoietic cells that engraft in 1° sheep recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the 20 recipient.

Isolation of non-hematopoietic stem cells from umbilical cord blood

An intrinsically pluripotent, CD45 negative population from placental cord blood, termed unrestricted somatic stem cells (USSC) as a rare population grows adherently and can be expanded to large quantities of cells without losing pluripotency. Thus, in vitro USSC homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, hematopoietic and neural cells including astrocytes and neurons expressing neurofilament, sodium channel protein and various neurotransmitter phenotypes are demonstrated. Stereotactic implantation of USSC into intact adult rat brain revealed that human Tau-positive cells persisted for several months, showing migratory activity and a typical neuron-like morphology. In vivo differentiation of USSC 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 USSC in a non-injury 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 as well as substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected still over one year following USSC transplantation. No malignant cell transformation was observed in any of these animals. An intrinsically pluripotent, CD45 negative population from placental cord blood, termed unrestricted somatic stem cells (USSC) as a rare population grows adherently and can be expanded to large quantities of cells without losing pluripotency. Thus, in vitro USSC homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, hematopoietic and neural cells including astrocytes and neurons expressing neurofilament, sodium channel protein and various neurotransmitter phenotypes are demonstrated. Stereotactic implantation of USSC into intact adult rat brain revealed that human Tau-positive cells persisted for several months, showing migratory activity and a typical neuron-like morphology. In vivo differentiation of USSC 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 USSC in a non-injury 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 as well as substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected still over one year following USSC transplantation. No malignant cell transformation was observed in any of these animals.

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 1015 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.

The Intracoelomic Route: A New Approach for in utero Human Cord Blood Stem Cell Transplantation

The intracoelomic route for in utero hematopoietic stem cell transplantation has been evaluated in pre-immune fetal sheep and the engraftment characteristics defined. Twelve ovine fetuses (gestational ages: 40–45 days) received intracoelomic transplants of human CD3-depleted (50 × 10⁶ per lamb) or CD34-selected (1–2 × 10⁵ per lamb) cord blood hematopoietic stem cells. Engraftment was evaluated from cell suspension of the liver, spleen, bone marrow and thymus by flow cytometry, cloning assays and polymerase chain reaction (PCR) analysis for human β₂-microglobulin gene. The engraftment of liver samples was also evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescent in situ hybridization (FISH) and immunohistochemistry. Four fetuses (33%) aborted shortly after intracoelomic transplantation and were not evaluable for engraftment. Engraftment was detected in 4 fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells. The degree of engraftment in these 4 fetuses ranged from 6 to 22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry). Three fetuses obtained after cesarean section at 102 (No. 435184) and 105 (Nos 915293, 037568) days and 1 fetus delivered at term, which received CD34-selected cord blood cells, had human engraftment with 10, 32, 20 and 10% CD45+ cells in bone marrow, respectively. A further check of human chimerism was done at 1 year after birth of the fetus delivered at term and 7.6% of bone marrow chimerism was detected. In 6 out of 8 fetuses evaluable for human engraftment, chimerism was confirmed by PCR analysis for human β₂-microglobulin which also identified human cells in brain, spinal cord, heart, lung and skeletal muscle. On liver samples, FISH and RT-PCR confirmed the xenograft of human cells and the immunohistochemical analysis detected human markers of hematopoietic and hepatic lineage of differentiation. This preliminary study indicates that intracoelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment.

A novel route of transplantation of human cord blood stem cells in preimmune fetal sheep: the intracelomic cavity.

The intracelomic route for in utero hematopoietic stem cell transplantation was evaluated in preimmune fetal sheep and the engraftment characteristics were defined. Twelve twin ovine fetuses (gestational age: 40-45 days) received intracelomic transplants of human CD3-depleted (50 x 10(6) per lamb) or CD34-selected (1-2 x 10(5) per lamb) cord blood hematopoietic stem cells. Engraftment was evaluated from cell suspensions of the liver, spleen, bone marrow, and thymus by flow cytometry, cloning assays, and polymerase chain reaction (PCR) analyses of human beta2-microglobulin. Four fetuses (33%) aborted shortly after intracelomic transplantation and were not evaluable for engraftment. Engraftment was detected in four fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells. The degrees of engraftment in these four fetuses ranged from 6%-22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry). Three fetuses obtained after cesarean section at 102 (no. 435184) and 105 (no. 915293, no. 037568) days and one fetus delivered at term that received CD34-selected cord blood cells had human engraftment with 10%, 32%, 20%, and 10% CD45(+) cells in bone marrow, respectively. In six of eight fetuses evaluable for human engraftment, chimerism was confirmed by PCR analysis for human beta2-microglobulin, which also identified human cells in brain, spinal cord, heart, lung, and skeletal muscle. This preliminary study indicates that intracelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment.

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

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. 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. 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. 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.

Human CD34- hematopoietic stem cells: basic features and clinical relevance.

CD34 has been widely used as a stem and progenitor cell marker, and clinical CD34+ stem cell transplantation (CD34+ SCT) has been performed for tumor purging and for prevention of graft-versus-host disease. Recently, CD34-negative hematopoietic stem cells (CD34 HSCs) were identified in mice and humans. Xenogeneic chimera engraftment assays with immunodeficient mice or preimmune fetal sheep resulted in identification of human CD34- HSCs in cord blood, bone marrow, and granulocyte colony-stimulating factor-mobilized peripheral blood, although no significant clonogenic activity was detected in vitro. These characteristics of CD34- HSCs make the assessment of clinical samples difficult. The generation of CD34+ HSCs from CD34 cells in vitro may be a surrogate assay for detecting CD34- HSC activity. This approach was used in recipients of CD34+ SCT and revealed the absence of a CD34 precursor population. The identification of a positive marker in CD34- HSCs and the development of a simpler and more efficient in vivo assay for CD34- HSCs may allow the diagnostic evaluation of human CD34- HSCs in various clinical procedures and diseases.