Objective-CD34 + cells, present within the bone marrow, have previously been shown to possess pancreatic endocrine potential. Based on this observation, we explored the capacity of CD34 + cells derived in culture from the differentiation of human embryonic stem cells (hESC), for their in-vivo pancreatic endocrine capacity.Methods-Sheep were transplanted with hESC-derived CD34 + cells, as well as non-sorted differentiated cultures. Transplantations were carried out with in-utero intraperitoneal injections prior to the development of the immune system in the fetus so that tolerance towards foreign antigens was acquired during gestation and persisted in the adult.Results-All cell populations that were tested demonstrated human cellular activity and long-term presence up to 5 years. However, the in-vivo beta-cell-like activity achieved from the transplantation of the sorted CD34 + cell population was not augmented by transplanting the entire cell population from which the CD34 + cells were isolated. Human DNA and insulin mRNA were detected in sheep pancreases. An average of 1.51 ng/mL human C-peptide was detected in serum from 8 animals transplanted with differentiated cell populations and assayed up to 55 months post-transplantation. Transplantation of as few as 23,500 cells resulted in long-term sustainable beta-cell like activity. Teratomas were absent in the transplanted animals.Conclusion-Our data suggest that hESC-derived CD34 + cells have a potential for long term invivo endocrine cellular activity which could prove useful in regenerative medicine. Since the same cell population has previously been shown to contain hematopoietic potential, it could be used for the induction of immunological tolerance and bone marrow chimerism prior to cellular therapy for diabetes. KeywordsHuman embryonic stem cells; Pancreas; Transplantation; Beta-cells; C-peptide Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. One potential therapy for diabetics is the restoration of beta-cell mass for the endogenous control of glycemia [1,2]. To this end, human embryonic stem cells (hESC) which have the capacity to form tissues of all three germ layers cells [3], are being evaluated for cellular transplantation therapy. hESC differentiation cultures have been developed for the derivation of progenitor cells that engraft and differentiate in-vivo into beta-cell-like cells [4,5]. Such research has had to occur in the absence of knowledge of a single or a group of cell-surface markers that may be used to unequivocally identify and isolate pancreatic stem cells [6]. Hence, focus has been placed ...
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: Engraftmentof allogeneic and xenogeneicHSC 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.
Bone marrow stromal cells (MSCs) represent a heterogeneous population derived from the non–blood-forming fraction of bone marrow that regulates hematopoietic cell development. In vitro, adult mesenchymal stem cells resident in this bone marrow fraction differentiate into bone, cartilage, and fat. Because MSCs can be easily obtained using a simple bone marrow aspiration and show extensive capacity for expansion in vitro, these cells have been considered as candidates for cell therapy. The aim of this study was to purify rat MSCs from adult bone marrow and to functionally characterise their abilities to differentiate along diverse lineages. Our data demonstrate that we successfully isolated, culture-expanded and differentiated a relatively homogeneous population of MPCs from adult rat bone marrow.
Objective: The purpose of this study was to assess the feasibility of in utero stem cell transplantation of human umbilical cord blood stem cells in fetal sheep and to compare two different techniques of in utero transplantation, namely ultrasound-guided in utero transplantation and in utero transplantation after midline celiotomy. Study design: Umbilical cord blood units were collected from term deliveries, after obtaining written informed consent. Human cord blood–derived, CD34+ stem cells were injected into the peritoneal cavity of 60- to 65-day-old ovine fetuses by using 2 different techniques: ultrasound-guided transabdominal percutaneous needle puncture and midline celiotomy with the exposure of the pregnant uterus. Engraftment was determined after birth by flow cytometry with use of human-specific anti-CD 34/45 antibodies. Results: We obtained a total of 3 chimeric lambs. Using the midline celiotomy technique the fetal loss rate was 75% and only 33,3% when using ultrasound-guided transabdominal percutaneous needle puncture technique. Engraftment of donor cells was found in all fetuses, with a mean level of 1.4% in fetal peripheral blood and 3.3% in fetal bone marrow. Conclusion: This preliminary study indicates that in utero stem cell transplantation of human hematopoietic cord blood stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment. We also concluded that the ultrasound-guided transabdominal percutaneous needle puncture technique is more effective than performing a midline celiotomy in terms of fetal loss rate.
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