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 donorderived 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. (HEPATOLOGY 2007;46:1935-1945 T he maintenance of cellular homeostasis within the normal liver and during liver regeneration is provided by mature hepatocytes and cholangiocytes and by the intrahepatic (IH) stem cell compartment located in the canals of Hering and the intralobular bile ducts. 1 Recently, another potential source of cells able to provide liver cell replacement has been identified. These stem/progenitor cells consist of hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) that reside within the bone marrow (BM) but can reach the liver through the circulatory system. [2][3][4][5][6][7][8][9][10][11][12] Because it is clearly evident that the shortage of available human donor organs cannot meet the needs of all the patients awaiting liver transplantation, alternatives to whole-organ replacement are urgently needed. Cell-based treatments, with cells of either hepatic or extrahepatic origin that would be able to repopulate and re-establish a functional liver after administration, could serve as a possible alternative to wholeorgan transplantation.In order to attain this goal, several questions, such as the sour...

SummaryThis study investigated whether relative changes that accompany the naturally occurring shifts in haematopoietic sites during human development play a role in haemoglobin (Hb) switching or whether Hb switching is innately programmed into cells. CD34 + /Lineage ) haematopoietic stem/progenitor cells (HSCs) were isolated from human fetal liver (F-LVR), cord blood (CB), and adult bone marrow (ABM), and the Hb was characterized by flow cytometry on cultures that generated enucleated red cells. All feeder layers (stroma from F-LVR, ABM, and human fetal aorta) enhanced cell proliferation and erythropoiesis but did not affect Hb type.HSCs from CB and F-LVR generated the same Hb profile under normoxia and hypoxia. HSCs from ABM had single-positive HbA and double-positive HbA and HbF cells at normoxia and almost entirely double-positive cells at hypoxia. Further characterization of these ABM cultures was determined by following mRNA expression for the transcription factors erythroid Kruppellike factor (EKLF) and fetal Kruppel-like factor (FKLF) as a function of time in cultures under hypoxia and normoxia. The erythroid-specific isoform of 5-amino-levulinate synthase (ALAS2) was also expressed under hypoxic conditions. We conclude that Hb switching is affected by the environment but not all HSCs are preprogrammed to respond.

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.

Objective-To determine if mesenchymal stem cells (MSC) derived from human fetal pancreatic tissue (pMSC) would engraft and differentiate in sheep pancreas following transplantation in utero.Methods-A three-step culture system was established for generating human fetal pMSC. Sheep fetuses were transplanted during the fetal transplant receptivity period with human pMSC and evaluated for in situ and functional engraftment in their pancreas, liver and bone marrow.Results-Isolation and expansion of adherent cells from the human fetal pancreas yielded a cell population with morphologic and phenotypic characteristics similar to MSC derived from bone marrow. This putative stem cell population could undergo multilineage differentiation in vitro. Three to 27 months after fetal transplantation, the pancreatic engraftment frequency (chimeric index) was 79% while functional engraftment was noted in 50% of transplanted sheep. Hepatic and marrow engraftment and expression was noted as well.Conclusion-We have established a procedure for isolation of human fetal pMSC that display characteristics similar to bone marrow derived MSC. In vivo results suggest the pMSC engraft, differentiate and secrete human insulin from the sheep pancreas.