Differentiation of encapsulated embryonic stem cells after transplantation.

BACKGROUND: Embryonic stem cells (ESC) when transplanted into recipients with different major histocompatibility antigens may be rejected, especially as cells differentiate and expression of these antigens increases. One method to prevent rejection is to place the developing ESC in microcapsules. It is currently unknown what effect encapsulation has on the ability of ESC to differentiate. METHODS: Human ESC (hESC; hES03 line) and mouse ESC (mESC; R1 line) were encapsulated in 2.2% barium alginate and transplanted intraperitoneally in SCID and BALB/c mice respectively. Cell morphology, viability, and gene characterization were assessed after retrieving the capsules up to four weeks from SCID mice and three months from BALB/c mice. RESULTS: Encapsulation prevented hESC and mESC from forming teratomas up to four weeks and three months, respectively. mESC but not hESC formed aggregates within the capsules, which remained free of fibrosis. Some but not all the transplanted encapsulated hESC differentiated towards all three lineages, but more so towards an endodermal lineage as shown by increased expression of alpha fetoprotein. This was similar to what occurred when encapsulated and non-encapsulated hESC were cultured in vitro for two weeks. In contrast to the hESC, transplanted encapsulated mESC differentiated mostly towards an ectodermal lineage as shown by increased expression of nestin and glial fibrillary acidic protein. In vitro, encapsulated and nonencapsulated mESC also began to differentiate, but not down any specific lineage. CONCLUSIONS: Encapsulated ESC do differentiate, although along multiple pathways, both when transplanted and maintained in culture, just as nonencapsulated ESC do when removed from their feeder layer.

Comparison of size, viability, and function of fetal pig islet-like cell clusters after digestion using collagenase or liberase.

Liberase is a highly purified blend of collagenases that has been specifically developed to eliminate the numerous problems associated with the conventional use of crude collagenase when isolating islet-like cell clusters (ICCs) from pancreases of different species. The influence of Liberase on yield, size, viability, and function of ICCs has been documented when this enzyme was used to digest adult but not fetal pancreases. In this study, we compared the effects of collagenase and Liberase on fetal pig ICCs. A total of eight fetal pig pancreas digestions were analyzed. Fetuses were obtained from Large White Landrace pigs of gestational age 80 +/- 2.1 days. The pancreases were digested with either 3 mg/ml collagenase P or 1.2 mg/ml Liberase HI. The time taken to digest the pancreas was shorter for collagenase when compared with Liberase (22 +/- 2 vs. 31 +/- 2 min). The size of ICCs was similar for both collagenase (83 +/- 0.5 microm) and Liberase (79 +/- 0.4 microm) as was the number of ICCs produced per pancreas (7,653 +/- 1,297 vs. 8,101 +/- 1,177). Viability, as assessed using fluorescent markers, was slightly greater for Liberase (79 +/- 1% vs. 76 +/- 1%, p < 0.05). Responsiveness to beta-cell stimulus (20 mM KCl) was similar for both methods of isolation, as was the insulin content of the ICCs, both in vitro and at I month after transplantation of 1,500 ICCs beneath the renal capsule of immunoincompetent mice. Despite the high content of endotoxins in collagenase, the above results show that this enzyme was equally as efficient as Liberase in isolating functional ICCs from fetal pig pancreas.

Effect of immunosuppressive doses of cyclosporine on pancreatic beta cell function in pigs.

OBJECTIVE: To evaluate whether immunosuppressive doses of cyclosporine (CsA) have an adverse effect on the liver, kidney, and pancreatic beta cells of pigs. ANIMALS: 8 juvenile 8-week-old Landrace X Large White crossbred pigs. PROCEDURE: CsA (100 to 140 mg/kg) was administered orally to euglycemic pigs to reach whole blood trough concentrations of approximately 1500 ng/mL. To determine pancreatic beta cell function, plasma C-peptide and insulin concentrations were measured in response to i.v. administration of glucose, glucagon, arginine, and oral administration of glucose. Effects on liver and kidney were determined by monitoring serum measurements of liver function and serum creatinine concentrations, respectively. RESULTS: Plasma concentrations of C-peptide were significantly lower in euglycemic CsA-treated pigs, compared with control pigs, following i.v. administration of glucose, glucagon, arginine, and oral administration of glucose. Furthermore, the glucose clearance rate was decreased in euglycemic CsA-treated pigs, compared with control pigs. Serum creatinine concentrations and 4 of 7 serum measurements of liver function were not adversely affected by CsA administration. Serum concentrations of bilirubin and albumin were significantly increased, and serum alanine aminotransferase activity was significantly decreased in CsA-treated pigs, compared with control pigs. Histologic evaluation of liver and kidney sections revealed no pathologic findings in CsA-treated or control pigs. CONCLUSIONS AND CLINICAL RELEVANCE: In our study, immunosuppressive doses of CsA caused an impairment of porcine pancreatic beta cell function, but did not have toxic effects on the kidney. However, on the basis of changes in serum bilirubin and albumin concentrations and alanine aminotransferase activity, subclinical toxic effects on the liver did occur when immunosuppressive doses of CsA were administered.

Neural precursors from canine skin: a new direction for testing autologous cell replacement in the brain.

Recent work indicates that neural progenitors can be isolated from the skin of rodents and humans. The persistence of these cells in accessible adult tissue raises the possibility of their exploitation for research and therapeutic purposes. This study reports on the derivation, culture, and characterization of homogenous canine skin-derived neuroprecursor cells (SKiNPs) from mature animals. Canine tissue was used because naturalistic brain diseases in community-dwelling dogs are emerging as ecologically sound models for a range of neurological conditions. Adult SKiNPs were initially isolated as neurospheres and then cultured for 10-15 passages in an adherent monolayer assay. Serumfree expansion conditions contained B-27, 20 ng/mL EGF, and 40 ng/mL bFGF. Gene expressions by PCR indicated expression of nestin, CD133, NCAM, and FGF2R, but not GFAP. Highly uniform expression of nestin (76 +/- 8.3%), NCAM (84 +/- 3.3%), betaIII-tubulin (96 +/- 4.3%), and CD133 (68 +/- 13.5%) was also observed. Directed differentiation of SKiNPs in the presence of serum induced betaIIItubulin, NSE, NCAM, and MAP2 in >90% of differentiated cells by immunophenotype analysis. Our culture system rapidly induces canine skin cells into neural precursors, maintains nestin expression in more than 75% of proliferating cells, and generates an almost universal neuronal-like phenotype after 7 days of in vitro differentiation. Their biological characteristics are suggestive of transiently amplifying fate-restricted neuroprecursors rather than true neural stem cells. This system may be an effective alternative for autologous neurorestorative cell replacement in canine models for further translational research.