Aug 012012
 

Introduction

Investigation of the origins of human brain disease has been limited by access to the organ and tissue of interest. The stunning pace of progress in the stem cell field over the last 10 years has overturned this barrier, with multiple possibilities for generating in vitro neurons from self-renewing cell sources. Multipotent neural stem cells (NSCs) isolated from fetal brain extracts were the first reported technique 1, and soon followed by derivation of pluripotent embryonic stem cells (ESCs) from the inner cell mass of the human blastocyst 2. Subsequently, NSCs and neural precursors were generated by transdifferention or redifferentation of somatic stem cell sources, including bone-marrow derived mesenchymal stem cellsand skin stem cells4,5. Most recently, there has been intense interest in the production of neurons from adult human fibroblasts through transgenic means – based on either the transduction of pluripotency genes to generate ESC-like induced pluripotential stem cells6 (iPS), or transduction of lineage-determining transcription factors to generate neurons directly (induced neuronal cells, iN7). The availability of neuronal culture methods based on a readily available patient skin biopsy has therefore heralded a dramatic rise in high-impact studies aimed at in vitro disease modeling of human brain disorders. This includes modeling of inherited brain diseases8,  as well as complex neuropsychiatric disorders such as schizophreniaand Alzheimer’s Disease10,11.

However, whilst generation of patient-specific neural stem cells (NSC) for the purpose of cell therapy is an exciting new experimental approach for the future treatment of neurodegenerative and neuropsychiatric disorders9-12, clinical translation is currently limited by two key factors:

  1. Efficient generation of sufficient quantities of a homogenous population of NSCs with fate-restricted neuronal differentiation capacity, and
  2. Current methods (e.g., human iPS) use of viral vectors to promote aberrant gene expression that is risky for human use11.

Accordingly, we have developed a protocol based on adult canine skin that can efficiently generate homogeneous populations of fate-restricted neural precursors without the use of any genetic manipulation5.

The purpose of this study is to adapt and optimise our successful adult canine skin protocol for use with human adult skin, and to comprehensively characterize the resultant cells.

This study will introduce a new technique for producing non-genetically modified neurons from human adult skin. This research may therefore have widespread significant applications in cell therapy, disease cell modeling, drug screening, clinical diagnosis and personalised medicine.

There are two main objectives for this study:

  • Develop and optimize a method for the induction, amplification and controlled differentiation of human adult SKiNPs.
  • Perform detailed characterisation of these cell lines using a wide range of gene-expression, protein, immunological, morphological, and functional assays

Skin-to-Neurons

 How we obtain our source of stem cells?

Patients undergoing elective orthopedic procedures are invited to participate in the study. After consent is obtained, a donated sliver of skin from the site of surgery is collected and quickly processed in the lab. Stem cells from the tissue sample are isolated and expanded. Afterwhich, the cells are either used to carry out various experiments or frozen down. The frozen cells are used to uphold a stock and build a library of human adult SKiNPs.

From Skin -To – Neurons: Techniques and Methods 

Immunocytochemistry (IHC):

Although antibody staining allows the visualization of neurons in culture, before, during and after the differentiation process, it falls short of showing distinct function of those cells. Therefore, staining is used in combination with other laboratory techniques, to help describe the structure, genetic expression and function of these derived neurons.

 

Human Adult SKiNPs on matrigel, express classical neural progenitor marker, Nestin (Red) along with the proliferative marker EdU (Green) 

 

 

 

 

 

 

 

Quantitative PCR: Quite surprisingly, Adult Human SKiNPs express the pluripotent marker Oct-4. This marker is usually expressed in embryonic stem cells, thus indicating to us, that these cells found in the adult, still have genetic characteristics of stemness. 

 

 

 

 

 

 

 

 

Electrophysiology: Patch-Clamp

 

Electrophysiology or patch-clamping will allow us to determine the functional properties of neurons derived from human adult SKiNPs. The patch-clamp method is currently the only technique that permits us to study in-depth the activity of single or multiple channels such as sodium and potassium channels. This is achieved by placing a glass microelectrode onto the membrane of the cell, encapsulating the channels of interest. The membrane is then gently “broken through,” forming a continuous closed circuit, or “whole cell” recording.

 

References

1          Reynolds, B. A., Tetzlaff, W. & Weiss, S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci12, 4565-4574 (1992).

2          Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science282, 1145-1147 (1998).

3          Jiang, Y. et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature418, 41-49 (2002).

4          Toma, J. G. et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol3, 778-784 (2001).

5          Valenzuela, M. J., Dean, S. K., Sachdev, P., Tuch, B. E. & Sidhu, K. S. Neural precursors from canine skin: a new direction for testing autologous cell replacement in the brain. Stem Cells Dev17, 1087-1094 (2008).

6          Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell126, 663-676 (2006).

7          Pang, Z. P. et al. Induction of human neuronal cells by defined transcription factors. Nature476, 220-223 (2011).

8          Park, I. H. et al. Disease-specific induced pluripotent stem cells. Cell134, 877-886 (2008).

9          Brennand, K. J. et al. Modelling schizophrenia using human induced pluripotent stem cells. Nature473, 221-225 (2011).

10        Qiang, L. et al. Directed conversion of Alzheimer’s disease patient skin fibroblasts into functional neurons. Cell146, 359-371 (2011).

11        Edelstein, M. L., Abedi, M. R. & Wixon, J. Gene therapy clinical trials worldwide to 2007 – an update. J Gene Med9, 833-842 (2007).

12        Valenzuela, M., Sidhu, K., Dean, S. & Sachdev, P. Neural stem cell therapy for neuropsychiatric disorders. Acta Neuropsychiatr19, 11-26 (2007).