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New protocol to ready clinical applications of induced pluripotent stem cells

Date:
April 3, 2013
Source:
New York Stem Cell Foundation
Summary:
Scientists have developed a new way to generate induced pluripotent stem cell lines from human fibroblasts, acquired from both healthy and diseased donors. This cell-sorting method consistently selects the highest quality, standardized iPS cells, representing a major step forward for drug discovery and the development of cell therapies.

Characterization of Fluorescence Activated Cell Sorted and Manually Derived IPSC Lines by Sendai virus. Immunofluroescence microscopy of the 1001.131.01 line demonstrating expression of common markers of pluripotency by FACS or Manually Derived IPSC lines. Nuclear Transcription Factors shown in Green, Surface Markers shown in Red, Nucleus stained with DAPI in Blue (A) Nanog/Tra-1-60 (B) Oct4/Tra-1-81 (C) Sox2/SSEA4 (D) Oct4/Alkaline Phosphatase. 10 Magnification
Credit: David J. Kahler et al. Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting. PLoS ONE, 2013; 8 (3): e59867 DOI: 10.1371/journal.pone.0059867

A team of New York Stem Cell Foundation (NYSCF) Research Institute scientists led by David Kahler, PhD, NYSCF Director of Laboratory Automation, have developed a new way to generate induced pluripotent stem (iPS) cell lines from human fibroblasts, acquired from both healthy and diseased donors. Reported in PLOS ONE, this cell-sorting method consistently selects the highest quality, standardized iPS cells, representing a major step forward for drug discovery and the development of cell therapies.

Employing a breakthrough method developed by 2012 Nobel laureate Shinya Yamanaka, MD, PhD, adult cells are "reprogrammed" or reverted to an embryonic-like state, commonly through viral infection. Reprogramming is a dynamic process, resulting in a mixture of fully reprogrammed iPS cells, partially reprogrammed cells, and residual adult cells. Previous protocols to select promising fully reprogrammed cells rely primarily on judging stem cell colonies by eye through a microscope.

Cell colonies selected by qualitative measures could include partially reprogrammed cells, a major concern for clinical applications of cell therapies because these cells could become any other cell type in a patient following transplantation. Additionally for drug efficacy assays and toxicity investigations on iPS cells, heterogeneous cell populations can mar the response of representative iPS cell lines.

The NYSCF scientists developed a quantitative protocol, optimized over three and a half years, in order to consistently harvest early-reprogrammed cells. Using fluorescence activated cell sorting (FACS), fully reprogrammed cells were identified by two specific proteins, or pluripotency markers. The group then looked at third marker that is expressed by partially reprogrammed or adult cells, and they then negatively selected against these cells to obtain only fully reprogrammed cells.

"To date, this protocol has enabled our group to derive (and characterize over) 228 individual iPS cell lines, representing one of the largest collections derived in a single lab," said Dr. Kahler. "This standardized method means that these iPS cells can be compared to one another, an essential step for the use in drug screens and the development of cell therapies."

This process of selecting stem cell colonies provides the basis for a new technology developed by NYSCF, The NYSCF Global Stem Cell Array (Array), a fully automated, robotic platform to generate cell lines in parallel. Currently underway at the NYSCF Laboratory, the Array reprograms thousands of healthy donors' and diseased patients' skin and/or blood samples into iPS cell lines. Sorting and characterizing cells at an early stage of reprogramming allows efficient development of iPS cell clones and derivation of adult cell types.

"We are enthusiastic about the promise this protocol holds to the field. As stem cells move towards the clinic, Dr. Kahler's work is a critical step to ensure safe, effective treatments for everyone," said Susan L. Solomon, CEO of NYSCF.


Story Source:

The above story is based on materials provided by New York Stem Cell Foundation. Note: Materials may be edited for content and length.


Journal Reference:

  1. David J. Kahler, Faizzan S. Ahmad, Anita Ritz, Haiqing Hua, Dorota N. Moroziewicz, Andrew A. Sproul, Carmen R. Dusenberry, Linshan Shang, Daniel Paull, Matthew Zimmer, Keren A. Weiss, Dieter Egli, Scott A. Noggle. Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting. PLoS ONE, 2013; 8 (3): e59867 DOI: 10.1371/journal.pone.0059867

Cite This Page:

New York Stem Cell Foundation. "New protocol to ready clinical applications of induced pluripotent stem cells." ScienceDaily. ScienceDaily, 3 April 2013. <www.sciencedaily.com/releases/2013/04/130403092655.htm>.
New York Stem Cell Foundation. (2013, April 3). New protocol to ready clinical applications of induced pluripotent stem cells. ScienceDaily. Retrieved July 28, 2014 from www.sciencedaily.com/releases/2013/04/130403092655.htm
New York Stem Cell Foundation. "New protocol to ready clinical applications of induced pluripotent stem cells." ScienceDaily. www.sciencedaily.com/releases/2013/04/130403092655.htm (accessed July 28, 2014).

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