The introduction of technologies capable of reprogramming human somatic cells into induced pluripotent stem (iPS) cells offers a unique opportunity to study many aspects of neurodegenerative diseases in vitro. This could ultimately lead to novel drug development and testing for these severe neurological disorders.
We recently reported that human dermal fibroblasts from a patient with relapsing-remitting Multiple Sclerosis (MS) were reprogrammed to pluripotency by retroviral transduction using defined factors (OCT4, SOX2, KLF4, and c-MYC). TheMSiPS cell lines resembled human embryonic stem (hES) cell-like colonies in morphology as well as gene expression and exhibited silencing of the retroviral transgenes after four passages.
The MSiPS cells were successfully differentiated into mature astrocytes, oligodendrocytes and neurons.
We have extended that study by generating additional iPS cell lines from MS patients displaying various stages of the disease and their healthy siblings as controls. The neural differentiation potential of those iPS cell lines was assessed.
In order to isolate and purify heterogeneous neural cell populations that were obtained from the differentiated MSiPS cell lines, a panel of antibodies was used to determine their surface markers signature.
Following two weeks of neural induction of MSiPS, we generated NSCs that could not only be propagated for many passages but could also be differentiated into a mixture of neurons and glial cells, as indicated by Immunofluoresence and PCR analysis.
The differentiated neural cells were subsequently sorted by FACS to obtain two distinct purified populations of neural cells, namely neurons and astrocytes. The neuron population was CD184-/CD44-/CD15 low /CD24+ and expressed mature and subtype specific neuronal markers. The astrocytes were CD184+/CD44+ and expressed the astrocyte marker GFAP.
This strategy provided us with highly purified population of neurons and glia cells, thus enabling downstream studies that require consistent and defined neural cell populations.
Current studies are underway to ascertain potential genetic and physiological differences that may exist between MS patients and their healthy sibling controls.