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

Abstract

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.

Dynamic expression of tRNA‐derived small RNAs define cellular states

“…Some of these transcripts are Klf4, Ina, Nr0b1, and Lrrc2, which are known pluripotency factors critical for the maintenance of the stem state [23]. Interestingly, we also observed an increase in tsGlnCTG association with pro-differentiation transcripts such as Myl3, CD44, and Dkk3 [24][25][26] (Fig 4D 0 and Dataset EV5). While initially counter intuitive, upon closer examination of these transcripts, we noticed a significant association (P value < 0.05) with genes involved in skeletal, cardiovascular, and blood vessel development compared with neuronal lineage (Dataset EV6).…”

Dynamic expression of tRNA-derived small RNAs define cellular states

“…Some of these transcripts include Klf4, Ina, Nr0b1 and Lrrc2, which are known pluripotency markers critical for the maintenance of the stem state (Sharova et al, 2007). However, we also observed an increase in tsGlnCTG association with pro-differentiation transcripts like Myl3, CD44 and Dkk3 (Cohen-Haguenauer et al, 1989;Quintanilla et al, 2014;Wang et al, 2015) (Fig. 3B' and Table S3).…”

Identification of differently expressed genes in leukemia using multiple microarray datasets

“…CD44 is a cell membrane glycoprotein that mediates the cellular response to the cellular microenvironment and regulates growth, survival, differentiation, and motility (Loh et al, 2014). CD44 is a negative cell surface marker of pluripotent stem cell identification during human fibroblast reprogramming (Quintanilla Jr. et al, 2014) and regulates endothelial cell proliferation and apoptosis by modulating CD31 and VE-cadherin expression (Tsuneki and Madri, 2014). A previous study suggested that CD44 contributed to lenalidomide resistance in multiple myeloma (Bjorklund et al, 2014).…”