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.

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.