Table 2.
The summary of the protocols for the differentiation of stem cells into the glomerular podocytes
| Original cells | hiPSCs | hiPSCs (Episomal iPS cell lines) | PGP1 hiPSCs cell line | CD34+ hHSCs | hAD-MSCs |
| Number of cells | NPHS1-GFP+ iPSCs (201B7) | 30,000/50,000 cells/cm2 | 4 × 104 cells/cm2 | 1 × 103 cells/cm2 | 5 × 105 cells/well of 6 well plate |
| Differentiation culture conditions (small molecules/growth factors/transcription factors/micro RNA) | iNPs aggregates, 0.8 µM polycarbonate filter, DMEM, 10% FCS, mouse embryonic spinal cord (E12.5), clone 3 on feeder free condition | Stepwise: ME, IM, NP, Podocytes. ME: Growth factor reduced matrigel coated dishes, For 3 days DMEM-F12, 2.5% FBS, GlutaMax (1:1), neurobasal media + N2B27 + CP21R7 (1 µM), BMP4 (25 ng/ml). IM: first medium replaced by STEMdiff APEL medium {RA (100 nM), BMP7 (50 ng/ml), FGF9 (200 ng/ml) for 2 days (total 5 days)}. NPs: 6th day for 7 days passaged by Accutase plated on type1 collagen-coated plates at a density of 20,000/40,000 cell/cm2 in VRAD medium {DMEM-F12 plus GlutaMax, 10% FBS, RA (80-100 µM), Vitamin D3 (100 nM)} | Stepwise: ME, IM, mature podocytes. On ECM (laminin 511-E8-coated plates). For 2 days in ME medium i.e. DMEM-F12 + GlutaMax, AA (100 ng/ml), CHIR99021 (3 µM), Y27632 (10 µM), 1X B27 serum free supplement. IM medium for 14 days DMEM-F12 + GlutaMax, BMP7 (100 ng/ml), CHIR99021 (3 µM), 1X B27 serum free supplement. Split cells 1:4 on ECM for 4-5 days in podocyte medium . DMEM-F12 + GlutaMax, BMP7 (100 ng/ml), CHIR99021 (3 µM), BMP7 (100 ng/ml), AA (100 ng/ml), VEGF (50 ng/ml), RA (0.1 µM), 1X B27 serum free supplement | Stepwise: For 5 days AA (10 ng/ml), RA (2.5-10 ng/ml, optimum 7.5 ng/ml), BMP7 (2.5-10 ng/ml, optimum 5 ng/ml) resulted OSR1+ cells. These cells for 9 days AA (10 ng/ml), RA (7.5 ng/ml), BMP7 (5 ng/ml), EGF (20 ng/ml), bFGF (20 ng/ml) | Stepwise: IM: for 3 days DMEM-F12, 2% FBS, AA (10 ng/ml), RA (10 µM). Three types of culture conditions + same basal medium 1. AA (10 ng/ml), RA (0.1 µM), BMP7 (20 ng/ml) 2. AA (10 ng/ml), RA (0.1 µM), GDNF (20 ng/ml) 3. AA (10 ng/ml), RA (0.1 µM), Wnt4 (50 ng/ml) |
| •Transfection by lipofectamine2000 | |||||
| •mi-RNA selection, miRNA-498 by TargetScan & Pictar algorithm | |||||
| Endpoint duration of analysis | Day 9 | ME day 2, IM day 4, NP day 6, mature podocytes day 13 | 21 days | 14 days | Day 9 |
| Detection methods/characterization | •ICC (WT1, E-CDH, CDH-6) (NPHS1, WT1, PODXL) | •ICC ME: (Oct4, T) IM: (Pax2, OSR1, LHX1) NP: (Pax2, Six2, WT1) | •Flow cytometry (Oct4, WT1, Nephrin) | •ICC (Podocin, SYNPO, GLEPP1), post 3 days (Pax2, WT1) post 9 days (Pax2, NPHS1, SULT1B1, NPHS2, SYNPO) | •ICC (OSR1, WT1, Pax2, Podocin, Nephrin, SYNPO, Laminin, HNA) |
| •IHC (H&E) Day 9 (Nephrin, GFP, WT1, Type IV collagen, E-CDH, CDH6, PODXL, CD31, human nuclear antibody) | •PCR ME: (T, Nanog) IM: (Pax2, OSR1, LHX1) NPs: (WT1, SYNPO, NPHS1, ACTN4, CD2AP, VEGF-A) | •ICC (Nephrin, WT1, Pax2, Podocin, Oct4, OSR1, EdU, PKCλ/I, Collagen type IV, FcRn receptor for albumin & IgG transport) | •Leishman’s staining | •Flow cytometry (OSR1) | |
| •Flow cytometry (Nephrin, PODXL) | •qRT-PCR (Six2, ACTN4, HPRT) | •qRT-PCR (POU5F1, Pax2, WT1, NPHS1, NPHS2) | •Western blot (Podocin, SYNPO) | •qRT-PCR (miR-498) | |
| •qRT-PCR (WT1, NPHS1, NPHS2, SYNPO, PODXL) | •SEM, day 6 | •Western blot (PKCλ/I) | •Flow cytometry (CD45, CD34) | •Western blot (Podocin, Nephrin, SYNPO, WT1) | |
| •Microarray | •SEM | •SEM | |||
| •SEM | |||||
| •TEM | |||||
| Functional analysis | •Transplantation of cells using solid agarose rods under NOD/SCID/JAK3 null mice kidney capsule, post 20 days characterized by | •Cell proliferation assay (differentiated vs. undifferentiated) | •ICC EdU-incorporation assay | •Tyrosine kinase assay | •Re-aggregation assay (E12.5) by HC, ICC (WT1, Laminin, Nephrin, HNA, Podocin) |
| •HC (H&E) | •Cytoskeleton rearrangement evaluation (peripheral localization of F-actin) | •Albumin uptake assay (confocal imaging & quantification of albumin positive cells) | •Scratch assay (1 × 106 cells on 0.8% agarose molds) | •Estimation of urinary protein excretion (Adriamycin induced-model in Balb/c mice) | |
| •IHC (WT1, CD31) | •Albumin uptake assay | •SEM | |||
| •Chimeric organoid cultures (E12.5 CD1 mouse) | |||||
| •ICC (E-CDH, HNA, WT1) | |||||
| References | [13] | [15] | [16] | [18] | [20] |