Improved Methods for Reprogramming Human Dermal Fibroblasts Using Fluorescence Activated Cell Sorting

Abstract

Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13NEGSSEA4POSTra-1-60POS on day 7–10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and “contaminating” partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.

Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells

“…Building upon our previous work 19 , we employed negative selection against incompletely reprogrammed cells with an immunomagnetic bead separation device (MACS) (Fig. 3a, Supplementary Fig.…”

“…To quantitatively assess pluripotency, we automated the spontaneous differentiation of EBs in V-bottom plates and performed a modified version of the previously described stem cell "scorecard" gene expression assay 19,20 to measure propensity for differentiation into the embryonic germ layers (ectoderm, mesoderm and endoderm) relative to hESC EBs ( Fig. 4a,b, Supplementary Fig.…”

“…The automated iPSC sorting method was based on a FACS method we had previously developed 19 . The worklist defined the 24-well source plate to be sorted and the 96-well destination plates that the sorted iPSCs should be seeded into.…”

A scalable label-free approach to separate human pluripotent cells from differentiated derivatives

“…For the hESC/ HDF separation experiments, HDF were discriminated from hESCs using an antibody to CD13 (APC-conjugated mouse anti-human CD13, BD Pharmingen TM ; 1:1000 dilution) (Kahler et al, 2013) and an appropriate isotype control. Depletion calculations were based on changes in numbers of CD13-positive cells in the various cell samples (feed, permeate, and retentate) following separations.…”

Strategies for Oral Mucosal Repair by Engineering 3D Tissues with Pluripotent Stem Cells

“…7 More recently, small molecules have been used as an alternative method for reprogramming in the hope of avoiding complications linked to delivering reprogramming factors using viral vectors. Zhang et al 9 and Kahler et al 10 Oral tissues have been shown to be a very useful source of cells that can be targeted for reprogramming to iPSC. Dental pulp stem cells, stem cells from exfoliated deciduous teeth, stem cells from apical papilla, as well as oral mucosal fibroblasts, have all shown a high efficiency of reprogrammed to iPSC.…”