Journal: International Journal of Molecular Sciences

Article Title: Molecular Determinants of the Human Retinal Pigment Epithelium Cell Fate and Potential Pharmacogenomic Targets for Precision Medicine

doi: 10.3390/ijms26125817

Figure Lengend Snippet: Phenotyping of hiPSCs. ( A ) Time-course imaging of reprogrammed HSC CD34+ cells showing pre-iPSC colonies emerging during the first three weeks. Trophoblast-looking structures detectable for some clones (*). Images acquired in brightfield (4×, 10×, 20×, 40×) (e.g., D63 P7) and phase contrast (10, 20×, 40×) (e.g., d101, P16) with an inverted phase contrast Nikon TS100. Scale bar is 50 μm. ( B ) Pre-iPSCs in naïve state form rosettes (black arrowheads) between passages 4 and 7 (P4–P7). The lumen (red arrowheads) emerges afterwards, until passage 12 (P7–P12) towards a primed iPSC state. ( C ) Phenotypic characterization of presumptive hiPSCs by qRT-PCR and immunocytochemistry, with analysis for canonical stem cell markers. Expression fold change in iPSC-derivative cell lines from three donors across passages (one bar per donor) (0–4 months) relative to control (peripheral blood mononuclear cells prior to purification, 72 h before reprogramming) featuring mRNA expression for c-KIT, REX, TERT, UTF1, CDH1, SOX2, l-MYC, OCT4, NANOG, PAX6, Lin28A, and DNMT3b (GAPDH, housekeeping gene). Box-whiskers features log2 fold change (median in horizontal bars, mean as a cross) in hiPSCs relative to PBMC controls. Fold change is 2 (−∆∆Ct) . Bilateral t-student test (hiPSCs vs. PBMC): |Stat-t| ≥ T α/2 on ∆∆Ct without error propagation. c-KIT (don1, df = 13, p -value = 0.0032; don2, df = 6, p -value = 0.0026; don3, df = 8, p -value = 0.0018), TERT (don1, df = 7, p -value = 0.0005 including pre-iPSCs clones; don2, df = 6, p -value = 5.73E-05; don3, df = 8, p -value = 0.015). CDH1 (pre-iPSCs colonies excluded; don1, df = 9, p -value = 0.006; don2, df = 6, p -value= 0.0004). SOX2 (don2, df = 8, p -value = 0.045); OCT4 (don1, df = 15, p -value = 3.39 × 10 −5 ; don2, df = 8, p -value = 0.0001; don3, df = 10, p -value = 0.0044); NANOG (don1, df = 15, p -value = 0.0003; don3, df= 10, p -value = 0.0006). PAX6 before passage 6 (don1, df = 9, p -value = 0.009; don2, df = 4, p -value = 0.0189; don3, df = 4, p -value = 0.043). LIN28A (don1, df = 15, p -value = 0.0002; don2, df = 8, p -value = 0.0046; don3, df = 10, p -value = 0.0003), DNMT3 (don1, df = 14, p -value = 3.69E-05; don2, df = 8, p -value = 2.43E-06; don3, df= 10, p -value = 0.0023). * applies if p -value < 0.05; ** if p -value < 0.01; *** if p -value < 0.001. Representative image of early neuro-ectodermal progenitors at 3 months from iPSCs derivation is displayed. Cells are stained for NESTIN and OCT-4. Nuclei are counterstained with Hoechst 33258. Confocal image displayed on maximum intensity projection (Zeiss LSM880). Scale bar is 40 μm.

Article Snippet: Following density gradient centrifugation of whole blood and retention of PBMCs, immunomagnetic purification of HSCs was performed according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany, CD34 MicroBead Kit UltraPure, human), and cell eluates were assessed by flow cytometry with CD45 (BD PharmingenTM, Milpitas, CA, USA, 560274), CD34 (Miltenyi Biotec, Bergisch Gladbach, Germany, 130-120-520), and CD90 (Miltenyi Biotec, Bergisch Gladbach, Germany, 130-117-796).

Techniques: Imaging, Clone Assay, Quantitative RT-PCR, Immunocytochemistry, Expressing, Control, Purification, IF-P, Staining

Journal: International Journal of Molecular Sciences

Article Title: Molecular Determinants of the Human Retinal Pigment Epithelium Cell Fate and Potential Pharmacogenomic Targets for Precision Medicine

doi: 10.3390/ijms26125817

Figure Lengend Snippet: Generation of hiPSC-iRPE . ( A ) iPSC cells express markers of stemness and neuroepithelial progenitors. iPSCs rosettes were identified by immunostaining for Tra-1–60, NANOG, VE-cadherin, and SSEA-5 between the second and third months of expansion (maximum intensity projection, Zeiss LSM880. Scale bar is 40 μm), passaged until exhaustion of episomal reprogramming vectors until P10, and retained for iRPE differentiation (see methods). Self-renewing, neuroepithelial-like stem cells were stained with Nestin and Oct4 and counterstained with Hoechst 33258 on their third month of iPSC expansion prior to iRPE induction. The emergence of RPE-primed neuroectoderm, differentiating RPE and induced RPE tissue patches over semi-permeable transwells was monitored over a period of 7 months from RPE induction. ( B ) Stratified differentiative niches and pigmented halos emerge during iRPE genesis. Differentiative niches were monitored over time by brightfield microscopy (4×, 10×, 20×, 40×) and phase contrast (10×, 20×, 40×), leading to fully pigmented, stereotypically cuboidal RPE beehive monolayers after two months. Images acquired with Nikon TS100. Scale bar is 50 μm. Induced iPSCs transition fromconcentric colonies to a layer of disorganized yet evenly distributed cells across the well and, within a month from neuro-epithelial induction, they acquire a cuboidal, globular or fusiform morphology, characteristic of presumptive RPE. hiPSCs cells are interspersed with differentiative niches that are constrained by fibrotic, concentric structures. Differentiated RPE can be retained upon subsequent but limited passages before de-differentiation or phenotypic EMT switch occurs (see methods). ( C ) Bipotent, neuroepithelial progenitors give rise to presumptive RPE, following a hiPSCs mesenchymal-epithelial transition and priming, and neurons. Comparison of three different healthy donor-derived hiPSCs indicates differences in the propensity to generate RPE. hiPSCs derived from three healthy donors. Neuro-ectodermal progenitors were stained after 10 days of exposure to RPECM, 4 days in RPEMM, 10 days in RPEMM (+PGE2), and at three months from induction leading to RPE-primed neuro-epithelial progenitors, immature RPE, and terminally differentiated RPE or alternate neuro-epithelial derivatives. ( D ) Representative screenshot of functionally tested, fully differentiated hiRPE tissue samples (D80) subject to RNA-seq. FPKMs are averaged across transcripts for any given gene. RPE progenitor markers include MITF, PMEL17, and TYRP1. Differentiated RPE markers include RPE65, DCT, BEST1, GPNMB, ALDH1A3, SERPINF1, AQP1, and CST3. FPKM values for transcript ENST00000262340 (RPE65) are 101.67 and 72.7 for terminally differentiated RPE, 3.5 and 1.7 for RPE progenitors, and undetectable in hIPSCs samples.

Article Snippet: Following density gradient centrifugation of whole blood and retention of PBMCs, immunomagnetic purification of HSCs was performed according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany, CD34 MicroBead Kit UltraPure, human), and cell eluates were assessed by flow cytometry with CD45 (BD PharmingenTM, Milpitas, CA, USA, 560274), CD34 (Miltenyi Biotec, Bergisch Gladbach, Germany, 130-120-520), and CD90 (Miltenyi Biotec, Bergisch Gladbach, Germany, 130-117-796).

Techniques: Immunostaining, Staining, Microscopy, Comparison, Derivative Assay, RNA Sequencing