Making Stem Cell Lines Suitable for Transplantation

Abstract

Human stem cells, progenitor cells, and cell lines have been derived from embryonic, fetal, and adult sources in the search for graft tissue suitable for the treatment of CNS disorders. An increasing number of experimental studies have shown that grafts from several sources survive, differentiate into distinct cell types, and exert positive functional effects in experimental animal models, but little attention has been given to developing cells under conditions of good manufacturing practice (GMP) that can be scaled up for mass treatment. The capacity for continued division of stem cells in culture offers the opportunity to expand their production to meet the widespread clinical demands posed by neurodegenerative diseases. However, maintaining stem cell division in culture long term, while ensuring differentiation after transplantation, requires genetic and/ or oncogenetic manipulations, which may affect the genetic stability and in vivo survival of cells. This review outlines the stages, selection criteria, problems, and ultimately the successes arising in the development of conditionally immortal clinical grade stem cell lines, which divide in vitro, differentiate in vivo, and exert positive functional effects. These processes are specifically exemplified by the murine MHP36 cell line, conditionally immortalized by a temperature-sensitive mutant of the SV40 large T antigen, and cell lines transfected with the c-myc protein fused with a mutated estrogen receptor (c-mycER TAM), regulated by a tamoxifen metabolite, but the issues raised are common to all routes for the development of effective clinical grade cells.

Islet neogenesis from the constitutively nestin expressing human umbilical cord matrix derived mesenchymal stem cells

“…Mice transplanted with ILCs from hUCMSCs into Kidney capsules showed the reversal of hyperglycemia without any signs of immune rejections of graft and teratomas formation which is in accordance with earlier reports. 1,13,14,31,32 However, the histopathology of transplanted kidney graft showed disintegration of ILCs to mesenchymal cells (data not shown). Hence we have later used immunoisolatory biocompatible capsules for the transplantation of ILCs generated from human tissue into mice (xenotransplantation).…”

Methodological study investigating long term laser Doppler measured cerebral blood flow changes in a permanently occluded rat stroke model

“…The CTX0E03 cells were provided by ReNeuron Ltd., Guildford, UK, created under the conditions described by Hodges et al (2007) and used for transplantation in this study. Briefly, the cells were revived from cryopreservation, plated on laminin-coated flasks in medium as described previously (Pollock et al, 2006) and grown at 37 • C, 5% CO 2 to 80% confluence before dissociation with TrypZean/EDTA [Cambrex] and Trituration solution (0.55 mg/ml Trypsin inhibitor [Sigma], 1% HSA, 25 U/ml Benzonase [Merck] in DMEM:F12) to neutralize the TrypZean and digest naked DNA.…”

Human Neural Stem Cell Therapy for Chronic Ischemic Stroke: Charting Progress from Laboratory to Patients

“…Neurospheres, for example, may contain mixed populations of stem cells and progenitors at differing stages of development, which may prevent the expansion of a homogenous cell line and limit their clinical potential [18]. In contrast, genetically immortalized NSCs, using genes for transcription factors such as Myc, have proved to be highly effective in extending the life span of hNSCs in vitro and in maintaining a stable genotype and phenotype [19,20].…”

“…1) [10,18,23]. CTX is genetically modified by the insertion of a single copy of the c- myc gene fused with a modified murine estrogen receptor (ER) [18,23,24]. The transgene expresses a recombinant protein (c-MycER TAM ), which is present as an inactive monomer in the cytosol of the cell [24].…”