CD44 Is a Negative Cell Surface Marker for Pluripotent Stem Cell Identification during Human Fibroblast Reprogramming

<div><p>Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones.</p></div

CD44 expression is gradually lost during fibroblast reprogramming.

<p>Flow cytometry dot plots with CD44-Alexa Fluor® 488 signal on the x-axis and SSEA4-Alexa Fluor® 647 signal on the y-axis. Lines demarcate quadrants of negative and positive signals for the two fluorophores, and the numbers at each corner indicate the percentage of cells per quadrant. The data compare (A) parental BJ fibroblasts, (B) H9 ESCs, (C) Day 9 reprogramming samples, and (D) Day 26 reprogramming samples.</p

CD44 is a positive fibroblast marker and a negative PSC marker.

<p>(A) CD44 immunostaining of (i) MEFs, (ii) BJ fibroblasts, (iii) feeder-free H9 ESCs, (iv) feeder-free iPSCs, (v) H9 ESCs on MEF feeders, and (vi) iPSCs on MEF feeders. The merged images shown consist of phase contrast and CD44 signal (green) (Scale bar: 200 µm). (B) Flow cytometry histograms of CD44-Alexa Fluor® 488 signal intensity in stained samples (solid black line) and unstained samples (dotted gray line) of (i) MEFs, (ii) BJ fibroblasts, (iii) feeder-free H9 ESCs, (iv) feeder-free iPSCs, (v) H9 ESCs on MEF feeders, and (vi) iPSCs on MEF feeders. (FF  =  feeder-free).</p

Description of parental BJ fibroblasts, control pluripotent H9 ESCs and episomal iPSCs, and iPSC clones used in the study.

<p>ND  =  Not determined.</p

Fully reprogrammed and partially reprogrammed clones are distinguished by combining multiple methods of characterization.

<p>(A) iPSC clones generated from BJ fibroblasts and characterized for the presence of pluripotent markers AP, SSEA4 and TRA-1-60. Figure shows phase contrast and fluorescence images merged together (Scale bar: 200 µm). (B) Principal Component Analysis of the global gene expression data from the controls and the iPSCs generated in the study. The three major clusters are demarcated by the red, green, and dark blue boxes. The dark blue and light blue boxes indicate the same clones, but at P5 and P16/17, respectively. (C) Pluripotency scores obtained using PluriTest^TM analysis of the global gene expression data for the cells used in the study. The area marked by red lines depicts the region under which 95% of pluripotent samples are expected to fall, while the region between the blue lines depicts where 95% of non-pluripotent samples fall. The dark blue and light blue boxes again indicate the same clones, but at early and late passages, respectively. (D) Immunostaining of trilineage differentiation markers AFP, βIIITub and SMA (red) in Day 21 differentiated ESCs and iPSCs used in the study (Scale bar: 200 µm).</p

CD44 is differentially expressed during human fibroblast reprogramming.

<p>(A) Venn diagram of genes coding for membrane proteins which were downregulated in H9 ESCs, fully reprogrammed iPSCs (FR iPSCs), and/or partially reprogrammed iPSCs (PR iPSCs) compared to BJ fibroblasts according to the global gene expression analysis. (B) Bar graph of microarray signal intensity plotted on the y-axis for each gene for BJ fibroblasts (white bars, n = 2), partially reprogrammed cells (black bars, n = 2) and iPSC clones (gray bars, n = 8). (C) Microarray-detected expression levels of <i>ACTB</i> (gray bars) and <i>CD44</i> (black bars) plotted on the y-axis for BJ fibroblasts and pluripotent cell types, represented by H9 ESCs cultured with feeders (n = 2), feeder-free (FF) H9 ESCs (n = 2), iPSCs with feeders (n = 6) and feeder-free (FF) iPSCs (n = 2). For the graphs, the error bars represent standard error of the mean. * indicates p-values <0.05, ** marks p-values <0.005, and *** signifies p-values <0.0005 when compared to BJ fibroblasts in an ANOVA analysis.</p

CD44 and SSEA4 co-staining distinguishes three types of colonies.

<p>Immunostaining of two independent reprogramming experiments at 23(i) and 26 days (ii) after transduction using antibodies against SSEA4 (magenta), and CD44 (green). The merged panel provides an overlay of the phase contrast image with both fluorescence signals. Both reprogramming experiments yield (A) CD44^negative SSEA4^positive colonies, (B) heterogeneous colonies, and (C) CD44^positive SSEA4^positive colonies (Scale bar: 200 µm).</p

List of surface markers that are highly downregulated in H9 ESCs and fully reprogrammed cells (FR) compared to BJ fibroblasts but not in partially reprogrammed cells.

<p>List of surface markers that are highly downregulated in H9 ESCs and fully reprogrammed cells (FR) compared to BJ fibroblasts but not in partially reprogrammed cells.</p