Comparative Analysis of Different Media Condition for Growth & Maintenance of Induced Pluripotent Stem Cells

Background and Aim: The discovery of induced pluripotent stem cells(iPSCs) had a great impact on fundamental stem cell studies and diseasesmodeling; moreover, iPSCs have provided a new perspective on precisionand regenerative medicine. Accordingly, several types of xeno-free andfeeder-free media and matrixes were developed in the past decade tosupport culturing of iPSCs. All these media allow continuous growth ofpluripotent stem cells (PSCs) in an undifferentiated state representingpowerful tools to improve our understanding of disease mechanismsand develop therapies to treat these diseases. Currently, internationalstandards for the validation and characterization of PSCs are based onexpression of self-renewal and pluripotency markers. However, in ourview there is a lack of in-depth molecular information regarding the truecharacter and uniformity of PSCs.Methods: To do this, we are reprogramming three different human healthydermal fibroblasts using CytoTune iPS 2.0 Sendai Reprogramming Kit.Next, transduced fibroblasts will be plated on different matrixes and feedwith different feeder & feeder-free media. After the emergence of iPScolonies, four colonies from each condition will be picked using TRA-1-60 Kit for Live Cell Imaging and propagated till passage 8 and willcharacterize according to general characterizations standard. To gainmore insight into differences or similarities between xeno-free and feederfreemedia, we will perform comparative transcriptome and methylomeanalysis of three clones per each fibroblast line and reprogrammedand grown under different media conditions selected based on theircharacterization and absence of Sendai virus. We recently developed asimple but thorough method to study DNA methylation in detail (Boerset al, 2018). This MeD-seq technology is based on a methyl-dependentrestriction enzyme LpnPI, which recognizes 50% of all methylated CpGs,and generates 32bp fragments for sequencing. LpnPI activity is blockedby a short template size preventing over-digestion of densely methylatedregions.Results: We recently applied MeD-seq to compare fully and partiallyreprogrammed iPSCs, and using unsupervised clustering easily clusterthe partially reprogrammed iPSCs apart from fully reprogrammed iPSCs.This underscores the power of this new technology that will now beapplied in conjunction with RNA sequencing to interrogate effects ofdifferent media conditions on the epigenome and transcriptome.Conclusion: This study might shed light on the quality of each culture condition and its consequent ability to generate stem cells suitable foruse in regenerative medicine and disease modeling.

کلیدواژه ها:

Reprogramming ، Induced Pluripotent stem cells ، LpnPI ، MeDseq