Sophia K. Dean

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 μm) and Liberase (79 ± 0.4 μm) as was the number of ICCs produced per pancreas (7653 ± 1297 vs. 8101 ± 1177). Viability, as assessed using fluorescent markers, was slightly greater for Liberase (79 ± 1% vs. 76 ± 1%, p < 0.05). Responsiveness to β-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 1 month after transplantation of 1500 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.

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.

Neural stem cell therapy for neuropsychiatric disorders

Objective: To conduct a comprehensive literature review of the area of neural stem cells and neuropsychiatry. Methods: ‘Neural stem cells’ (NSCs) and ‘neurogenesis’ were used as keywords in Medline (1966 – November 2006) to identify relevant papers in the areas of Alzheimer’s disease (AD), depression, schizophrenia and Parkinson’s disease (PD). This list was supplemented with papers from reference lists of seminal reviews. Results: The concept of a ‘stem cell’ continues to evolve and is currently defined by operational criteria related to symmetrical renewal, multipotency and functional viability. In vivo adult mammalian neurogenesis occurs in discrete niches in the subventricular and subgranular zones – however, functional precursor cells can be generated in vitro from a wide variety of biological sources. Both artificial and physiological microenvironment is therefore critical to the characteristics and behaviour of neural precursors, and it is not straightforward how results from the laboratory can be extrapolated to the living organism. Transplant strategies in PD have shown that it is possible for primitive neural tissue to engraft into neuropathic brain areas, become biologically functional and lead to amelioration of clinical signs and symptoms. However, with long-term follow-up, significant problems related to intractable side-effects and potential neoplastic growth have been reported. These are therefore the potentials and pitfalls for NSC technology in neuropsychiatry. In AD, the physiology of amyloid precursor protein may directly interact with NSCs, and a role in memory function has been speculated. The role of endogenous neurogenesis has also been implicated in the etiology of depression. The significance of NSCs and neurogenesis for schizophrenia is still emerging. Conclusions: There are a number of technical and conceptual challenges ahead before the promise of NSCs can be harnessed for the understanding and treatment of neuropsychiatric disorders. Further research into fundamental NSC biology and how this interacts with the neuropsychiatric disease processes is required.

Alginate microcapsule as a 3D platform for propagation and differentiation of human embryonic stem cells (hESC) to different lineages.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche(1-3). The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment(4) such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures(2). These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded(5). Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions(2) to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes(1). We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, >100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

P2-470: Survival and differentiation of adult skin-derived neuroprecursors in a rat model of age-related cognitive impairment

mer s disease (AD). Up to now no resolutive pharmacological therapy exists and there are increasing arguments regarding the beneficial effect of natural nutrients, able to slow down the progression of the disease. Some have the ability to induce cell proliferation and/or survival. Adult neurogenesis could be a good therapeutic strategy for cognitive aging and neurodegenerative diseases(AD). The aims of this work has been to study the modulatory effect of *LMN diet (cocoa, nuts and other natural extracts) in adult mouse neurogenic brain areas. Methods: 129SV male mice, were feed during 40 days with a standard Harlan 2014 control diet and the same diet containing 9,27% of *LMN. Animals received BrdU injections. Brains were processed for inmunohistological and immunoblots studies. Results: Histological samples of mice feed with *LMN diet, showed a noticeable increasing number of proliferative cells by BrdU and PCNA staining, in the adult neurogenic areas: subventricular zone (SVZ) and subgranular layer of dentate gyrus (sgDG). Using Dcx and PSA-NCAM, an increasing in the undifferentiating neurons were determined in the granular layer of dentate gyrus and in the rostral migratory stream (RMS). The immature oligodendrocytes (Ng2) and astrocytes (GFAP) were also increasing in these brain areas. All these results were corroborated by Western-Blot analysis. Moreover, the several interneurons subpopulations of the olfactory bulb were changed. Increasing the tyrosine hydroxylase, calretinin and calbindin neuronal subpopulations, and decreasing the parvalbumin interneurons. In the dentate gyrus, the granular layer presented a high cell number when comparing with the control animals. Conclusions: All these findings showed that *LMN diet promotes the neurogenesis in the adult mouse neurogenic niches. The differences between interneurons subpopulations of olfactory bulb suggested that, *LMN diet could modulate differentiation process in the RMS. Therefore, *LMN diet could be a promising nutrient that would contribute to the neural replacement and to re-establish the brain function in order to avoid the cognitive decline, the main hallmark in Alzheimer’s disease. *Patent submitted: Reference ES2281270. Acknowledgements: This work has been financed by the Spanish Ministry of Industry, project INGENIO 2010CENIT ref MET-DEV-FUN (2006-2009).