Journal: Cells

Article Title: Efficient Generation of Pancreatic Progenitor Cells from Induced Pluripotent Stem Cells Derived from a Non-Invasive and Accessible Tissue Source—The Plucked Hair Follicle

doi: 10.3390/cells13121010

Figure Lengend Snippet: Cryopreserved hair follicle derived primary keratinocytes can be reprogrammed using a feeder-free system across multiple demographics. Expanded primary keratinocytes (5 × 10 5 cells) from 11 participants were taken for reprogramming at passages 2–3 ( A ). The cells were electroporated and reprogrammed using the Epi5 vector system and gradually placed into N2B27 media. At day 9, the media were switched to TeSR E7, after which the development of Embryonic Stem Cell (ESC)-like colonies was observed until day 21. The non-reprogrammed keratinocytes flattened and ceased to proliferate in culture. Scale bar = 100 µm and 200 µm for d33 ( B ). All 11 participants were able to generate at least one colony, which could be further expanded by day 21 ( C ). In total, 4 participant cell lines from the older (>60) and younger demographic (<40) were further expanded, and episome-free lines were generated. All the cell lines exhibited ESC morphology. Scale bar = ( D ). The reprogrammed participants’ ages were correlated to their reprogramming efficiency (# of colonies formed on day 21). There was no significant difference observed ( E ). There was also no significant difference between reprogramming efficiency and sex, with both sexes having similar colony-forming units. (ns: non-significant.) ( F ). There was a significant positive correlation when comparing reprogramming efficiency and % outgrowth success (# of follicles with outgrowth/10) ( G ).

Article Snippet: In total, 1 μL of Epi5 Reprogramming Vectors (Oct4, Sox2, Klf4, L-Myc, and Lin28) and 1 μL of Epi5 p53 and EBNA vectors (Life Technologies, Carlsbad, CA, USA) were added to each 100 μL solution.

Techniques: Derivative Assay, Plasmid Preparation, Generated

Journal: Cells

Article Title: Efficient Generation of Pancreatic Progenitor Cells from Induced Pluripotent Stem Cells Derived from a Non-Invasive and Accessible Tissue Source—The Plucked Hair Follicle

doi: 10.3390/cells13121010

Figure Lengend Snippet: Induced pluripotent stem cells derived from cryopreserved hair follicles maintain pluripotency in vitro and are genetically stable. The episome-free lines were evaluated for pluripotency marker expression using immunofluorescence and confocal imaging. All the cell lines were positive for OCT4 (Green = FITC488]), Nanog (Green = FITC488), and SOX2 (Red = CY5647), as represented by cell line N3. Scale Bars = 200 μm ( A ). The cells were then expanded at various passages and cell population doublings (CPD), and were analyzed using G-banding analysis. Routine G-banding analysis was carried out, and 15 metaphases per cell line were examined. All four cell lines had no major chromosomal abnormalities, and the lines exhibited their respective XY: XX chromosome patterning ( B ).

Article Snippet: In total, 1 μL of Epi5 Reprogramming Vectors (Oct4, Sox2, Klf4, L-Myc, and Lin28) and 1 μL of Epi5 p53 and EBNA vectors (Life Technologies, Carlsbad, CA, USA) were added to each 100 μL solution.

Techniques: Derivative Assay, In Vitro, Marker, Expressing, Immunofluorescence, Imaging

Journal: PLoS ONE

Article Title: A Universal System for Highly Efficient Cardiac Differentiation of Human Induced Pluripotent Stem Cells That Eliminates Interline Variability

doi: 10.1371/journal.pone.0018293

Figure Lengend Snippet: ( A ) Representative immunocytochemistry of pluripotency markers POU5F1 (OCT4), NANOG, TRA-1-81, and SSEA4 in hiPSC line CBiPSC6.2 after >20 passages. ( B ) Representative hematoxylin and eosin staining of teratoma sections derived from CBiPSC6.2 after >20 passages demonstrating ectodermal, endodermal and mesodermal lineage differentiation. All CBiPSC clones in these studies formed similar teratomas. ( C ) Real-time RT-PCR studies p15 CBiPSC lines for endogenous pluripotency genes using primers that distinguish endogenous expression from transgenes (see ). ( D ) The presence of plasmid transgene sequences examined by PCR at p11 in CBiPSC6.2, CBiPSC6.11, CBiPSC6.13, CBiPSC19.11 (lanes 3–6, respectively) and negative control H9 hESC (p48) (lane 1) compared to positive control early cultures from p2 (lane 2). RT-PCR analysis of selected plasmid sequences in p11 CBiPSC6.2, CBiPSC6.11, CBiPSC6.13, CBiPSC19.11 (lanes 8–11, respectively) and negative control H9 hESC (lane 7) and p2 cultures (lane 12). ( E ) Genomic Southern blot analysis for episomal vector backbone integration in lines CBiPSC6.2, CBiPSC6.11, CBiPSC6.13, CBiPSC19.11 (p15) (lanes 2–5, respectively), H9 hESC (p55) (lane 1). Combination 6 episomal vector DNA was diluted as positive control to the equivalents of 0.4 and 4 integrations per haploid genome (0.4× and 4×). L: 1 kb plus ladder. These studies were also conducted for non-viral adult fibroblast-derived hiPSC lines iPSCWT2 and iPSCWT4 with similar results (Machairaki et al. , in preparation).

Article Snippet: EBNA-based pCEP4 vectors pEP4 EO2S EN2L ( OCT4, SOX2, NANOG, LIN28 ), pEP4 EO2S ET2K ( OCT4, SOX2, SV40LT, KLF4 ), pEP4 EO2S EM2K ( OCT4, SOX2, MYC, KLF4 ) were obtained from Addgene (Cambridge, MA, http://www.addgene.org ).

Techniques: Immunocytochemistry, Staining, Derivative Assay, Clone Assay, Quantitative RT-PCR, Expressing, Plasmid Preparation, Negative Control, Positive Control, Reverse Transcription Polymerase Chain Reaction, Southern Blot