Hope Andrew
(Marc-Olivier Baradez, Cathy Oliveira, Kenny Pollock, John Sinden
ReNeuron Ltd, 10 Nugent Road, Surrey Research Park, Guildford, UK, GU2 7AF)
A c-MycERTAM conditionally immortalized neural stem cell line was derived from 12 week human fetal striatum. Cells were isolated by enzymatic digestion with trypsin in combination with mechanical trituration. Once established in culture these primary neural cells were transduced by retroviral with the c-MycERTAM transgene, integrated in chromosome 4, and a clonal population of cells isolated. The cells are maintained on laminin coated culture flasks using defined serum-free medium. Cell proliferation is conditionally dependent on the presence of mitogens (EGF and bFGF) and 4-hydroxytamoxifen (4-OHT), the ligand which activates the c-MycERTAM transgene (see Figure 1). The growth kinetics remain stable during banking of the cells through as many as 65 population doublings (see parallel lines in Figure 2), including a Master Cell Bank manufactured to GMP standards, and Drug Substance level banks, used for efficacy testing and preclinical development. The cells retain a normal male karyotype throughout this banking process (data not shown), indicating that they remain genetically stable.
Early evaluation of this cell line demonstrated efficacy in a rat model of Huntington’s Disease (HD; Abstract: Pollock et al. Neuroscience 2004, San Diego, USA.), where fine motor dysfunction is reversed.
More recently further characterisation of the cell line has been undertaken post GMP banking, including molecular and cellular phenotype evaluation. The transcript profile of STR0C05 has been assessed by quantitative RT-PCR (Figure 1) in comparison with 19 week human fetal brain RNA. The cells transcribe DARPP-32, doublecortin and other markers anticipated for striatal neuronal repair, as well as markers for glia and oligodendrocytes. Transcription of neural progenitor and stem cell markers demonstrate the immaturity of the culture.
Figure 1. Transcript profiling of STR0C05 cells. HD repair genes: PPP1R1B gene product is the striatal GABAergic MSN-specific DARPP-32; all others are present including doublecortin (DCX), expressed in migrating neurons. The mature neuron marker MAPT is not present, though glial and oligodendrocytic markers are (GFAP and CNP). PAX6 negativity confirms the striatal origin of STR0C05. Stem cell markers are also expressed (POU5F1 = Oct4). The transgene (Tg) is assayed through a mouse-specific stretch of the estrogen receptor in c-MycERTAM.
The neural multipotency of the cells is dependent upon the formation of neurons, glia and oligodendrocytes upon differentiation. The STR0C05 cells spontaneously differentiate when cultured in the absence of mitogens and 4-OHT. GFAP-immunoreactive astrocytes and GalC-immunoreactive oligodendrocytes form readily and can be detected by ICC within 7 days. Neurogenesis occurs following the formation of neural-rosettes (14 to 21 days), and neurons are immunoreactive for ?III-tubulin, and doublecortin (Figure 2).
In conclusion, STR0C05 is a c-MycERTAM immortalized clonal human neural stem cell line with efficacy in a rat model of HD. Augmented growth of the cells is conditionally dependent upon 4-OHT in the culture medium, under which conditions the cells are successfully manufactured and genetically stable. The molecular phenotype shows that the cells express genes involved in HD repair, and following differentiation they become neurons, glia and oligodendrocytes. These features recommend the cell line for preclinical development for the treatment of HD.
Figure 2. Differentiation of STR0C05 into neural phenotypes. Spontaneous generation of astrocytes (A; 14 days) and oligodendrocytes (B; 7 days) following mitogen and 4-OHT withdrawal (A, B; red = GFAP [astrocytes]; green = GalC [oligodendrocytes]). Following 14 days, neural-rosettes develop (C) from which neurons migrate (D; red = doublecortin; green = ?III tubulin). Control cultures grown in proliferating conditions (i.e. undifferentiated) are immunoreactive for the neural stem cell marker nestin (E; green = nestin).
Bars = 100 ?m. Blue = Hoescht counterstain.
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