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1.  Human Immune-reactivity against Liver Sinusoidal Endothelial Cells from GalTα(1,3)GalT-Deficient Pigs 
Cell transplantation  2010;19(6):783-789.
Elimination of galactose-α(1,3)galactose (Gal) expression in pig organs has been previously shown to prevent hyperacute xenograft rejection. However, naturally present antibodies to non-Gal epitopes activate endothelial cells leading to acute humoral xenograft rejection. Still, it is unknown whether xenogeneic pig liver sinusoidal endothelial cells (LSECs) from α(1,3)galactosyltransferase (GalT)-deficient pigs are damaged by antibody and complement-mediated mechanisms. The present study examined the xeno-antibody response of LSECs from (GalT)-deficient and wild pigs.
Isolated LSEC from wildtype and GalT pigs were expose to human and baboon sera, IgM and IgG binding was analyzed by flow cytometry. Complement activation (C3a and CH50) was quantified in vitro from serum-exposed LSEC cultures using Enzyme-Linked ImmunoSorbent Assay. Levels of complement activated cytotoxicity (CAC) were also determined by a fluorescent Live Dead Assay and by the quantification of LDH release.
IgM binding to GalT KO LSECs was significantly lower (80% human and 87% baboon) compare to wildtype pig LSEC. IgG binding was low all groups. Moreover, complement activation (C3a and CH50) levels released following exposure to human or baboon sera were importantly reduced (42% human and 52% baboon), CAC in GalT KO LSECs was reduced by 60% in human serum and by 72% in baboon serum when compared to wildtype LSECs and LDH release levels were reduced by 37% and 57% respectively.
LSECs from GalT KO pigs exhibit a significant protection to humoral-induced cell damage compare to LSECs from wild pigs when exposed to human serum. Though insufficient to inhibit xenogeneic reactivity completely, transgenic GalT KO expression on pig livers might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.
PMCID: PMC2957548  PMID: 20573304
Xenotransplantation; Liver endothelial cells; GalTα(1,3)GalT-Knockout pigs
2.  Activin Alters the Kinetics of Endoderm Induction in Embryonic Stem Cells Cultured on Collagen Gels 
Stem cells (Dayton, Ohio)  2007;26(2):474-484.
Embryonic stem cell-derived endoderm is critical for the development of cellular therapies for the treatment of disease such as diabetes, liver cirrhosis, or pulmonary emphysema. Here, we describe a novel approach to induce endoderm from mouse embryonic stem cells (mES) using fibronectin-coated collagen gels. This technique results in a homogenous endoderm-like cell population, demonstrating endoderm-specific gene and protein expression, which remains committed following in vivo transplantation. In this system, activin, normally an endoderm inducer caused an 80% decrease in the Foxa2 positive endoderm fraction, while follistatin increased the Foxa2 positive endoderm fraction to 78%. Our work suggests that activin delays the induction of endoderm through it transient precursors, the epiblast and mesendoderm. Long term differentiation, displays a two-fold reduction in hepatic gene expression and three-fold reduction in hepatic protein expression of activin-treated cells compared to follistatin-treated cells. Moreover, subcutaneous transplantation of activin-treated cells in a syngeneic mouse generated a heterogeneous teratoma-like mass, suggesting these were a more primitive population. In contrast, follistatin-treated cells resulted in an encapsulated epithelial-like mass, suggesting these cells remained committed to the endoderm lineage. In conclusion, we demonstrate a novel technique to induce the direct differentiation of endoderm from mES cells without cell sorting. In addition, our work suggests a new role for activin in induction of the precursors to endoderm, and a new endoderm-enrichment technique using follistatin.
PMCID: PMC2802581  PMID: 18065398
Activin; Endoderm; Collagen Gel; Embryonic Stem Cells (Mouse); Follistatin; Epiblast
Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to prevent the development of liver fibrosis in a number of pre-clinical studies. Marked changes in liver histopathology and serological markers of liver function have been observed without a clear understanding of the therapeutic mechanism by which stem cells act. We sought to determine if MSCs could modulate the activity of resident liver cells, specifically hepatic stellate cells (SCs) by paracrine mechanisms using indirect cocultures. Indirect coculture of MSCs and activated SCs led to a significant decrease in collagen deposition and proliferation, while inducing apoptosis of activated SCs. The molecular mechanisms underlying the modulation of SC activity by MSCs were examined. IL-6 secretion from activated SCs induced IL-10 secretion from MSCs, suggesting a dynamic response of MSCs to the SCs in the microenvironment. Blockade of MSC-derived IL-10 and TNF-α abolished the inhibitory effects of MSCs on SC proliferation and collagen synthesis. In addition, release of HGF by MSCs was responsible for the marked induction of apoptosis in SCs as determined by antibody-neutralization studies. These findings demonstrate that MSCs can modulate the function of activated SCs via paracrine mechanisms provide a plausible explanation for the protective role of MSCs in liver inflammation and fibrosis, which may also be relevant to other models of tissue fibrosis.
PMCID: PMC2096777  PMID: 17869217
stem cell; fibrosis; immunomodulation; liver injury
4.  Mesenchymal Stem Cell-Derived Molecules Reverse Fulminant Hepatic Failure 
PLoS ONE  2007;2(9):e941.
Modulation of the immune system may be a viable alternative in the treatment of fulminant hepatic failure (FHF) and can potentially eliminate the need for donor hepatocytes for cellular therapies. Multipotent bone marrow-derived mesenchymal stem cells (MSCs) have been shown to inhibit the function of various immune cells by undefined paracrine mediators in vitro. Yet, the therapeutic potential of MSC-derived molecules has not been tested in immunological conditions in vivo. Herein, we report that the administration of MSC-derived molecules in two clinically relevant forms-intravenous bolus of conditioned medium (MSC-CM) or extracorporeal perfusion with a bioreactor containing MSCs (MSC-EB)-can provide a significant survival benefit in rats undergoing FHF. We observed a cell mass-dependent reduction in mortality that was abolished at high cell numbers indicating a therapeutic window. Histopathological analysis of liver tissue after MSC-CM treatment showed dramatic reduction of panlobular leukocytic infiltrates, hepatocellular death and bile duct duplication. Furthermore, we demonstrate using computed tomography of adoptively transferred leukocytes that MSC-CM functionally diverts immune cells from the injured organ indicating that altered leukocyte migration by MSC-CM therapy may account for the absence of immune cells in liver tissue. Preliminary analysis of the MSC secretome using a protein array screen revealed a large fraction of chemotactic cytokines, or chemokines. When MSC-CM was fractionated based on heparin binding affinity, a known ligand for all chemokines, only the heparin-bound eluent reversed FHF indicating that the active components of MSC-CM reside in this fraction. These data provide the first experimental evidence of the medicinal use of MSC-derived molecules in the treatment of an inflammatory condition and support the role of chemokines and altered leukocyte migration as a novel therapeutic modality for FHF.
PMCID: PMC1978513  PMID: 17895982

Results 1-4 (4)