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Podocyte Differentiation from Nephron Progenitor Cells (Version 1.0) | ATLAS-D2K Center

PLEASE NOTE: ATLAS-D2K closed July 31, 2025 and this website is for reference purposes only.

Podocyte Differentiation from Nephron Progenitor Cells (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Uyen Tran; Michael Bukys; Jan Jensen; Oliver Wessely

Keywords

[‘embryonic stem cells (ESC)’, ‘hiPSC’, ‘nephrogenesis’, ‘Podocytes’]

Subjects

[‘Tissue culture’, ‘Developmental biology’]

Release Date

2019-11-27

Abstract

Using an unbiased, Design-of-Experiment (DOE) and data-driven approach towards stem cell differentiation, we have identified a combination of signaling agonists and/or antagonists that promote direct differentiation from human stem-cell derived nephron progenitors cells into podocytes.

Reagents

REAGENTS

  • Accutase, StemCell Technologies #07920
  • Advanced RPMI, Life Technologies #12633012
  • BGJ398 (FGF inhibitor), Selleckchem #S2183
  • Chemically Defined Lipid Concentrate, Gibco #11905-031
  • CHIR99021 (CHIR), LC Laboratories #C-6556
  • Geltrex, ThermoFisher #A1413302
  • Glutamax, Life Technologies #35050-061
  • Insulin, Roche #1376497
  • IWP2, Selleckchem #S7085
  • Laminin-521, Biolamina #LN521-02
  • LDN193189, StemCell Technologies #72142
  • Lysophosphatidic Acid (LPA), Santa Cruz Biotech #325465-93-8
  • Monothioglycerol, Sigma #M6145
  • Nephron Progenitor Cells prepared using the protocol developed by Morizane et al., 2015 (see Note 1)
  • Noggin, Peprotech #120-10C
  • Polyvinyl Alcohol, Sigma #P8136
  • Recombinant Activin A, Peprotech #120-14
  • Recombinant Bmp4, Peprotech #120-05ET
  • Recombinant Bmp7, Peprotech #120-03
  • Recombinant Fgf9, R&D Systems #273-F9-025
  • Transferrin, Roche #652202
  • XX (gamma-Secretase inhibitor), Millipore/Sigma #565789

CELL CULTURE MEDIA

Advanced RPMI/Glutamax Mix 1 ml Glutamax with 99 ml Advanced RPMI and filter. Media is good for at least 2 weeks.

CDM2 [Loh et al. (2014)] 50% IMDM 50% F12 1mg/ml Polyvinyl Alcohol 1% v/v Chemically Defined Lipid Concentrate 450 uM Monothioglycerol 0.7 ug/ml Insulin 15 ug/ml Transferrin

Preparation:

  1. Prepare Insulin stock solution. 100 mg insulin crystalline powder is dissolved in 10 ml of double distilled autoclaved water (final concentration of 10 mg/ml)
  2. Prepare media supplement. 70 ul of insulin (10 mg/ml) 500 ul of transferrin 10 ml of chemically defined lipid concentrate 39 ul of Monothioglycerol Aliquot 1,060.9 ul/tube and store at -20⁰C. One aliquot of supplements is used to prepare 100 ml of CDM2.
  3. Prepare CDM2 (100 ml)
    • Thaw one aliquot of the media supplement.
    • 100 mg PVA is weighed out and placed into a beaker. Add 50 mL of IMDM media and stir until dissolve.
    • 50 ml of F12 are added to the top of a bottle top filter.
    • The previously prepared IMDM/PVA tube is also then added to the top of a bottle top filter.
    • The thawed insulin/transferrin/lipid/monothioglycerol aliquot is added to the top of a bottle top filter.
    • If penicillin and streptomycin is needed in the medium add 1 ml of a 1/100 solution to the top of a bottle top filter.
    • Add 1 mL of L-glutamine.
    • Vacuum filter, remove the bottle top filter and label with date.
    • CDM2 media is good for about 2-3 weeks.

Podocyte Matrix (always prepare fresh before you add it) 25 nM FGF inhibitor 30 ng/mL BMP4 30 ng/mL BMP7 1 uM LPA 100 nM IWP2 100 nM XX inhibitor (gamma-secretase inhibitor) 125 nM LDN193189 in either Advanced RPMI/Glutamax or CDM2 (see Note 5)

Procedure

Day 1 We use nephron progenitor cells prepared following the protocol published by Morizane et al. (2015) (see Notes 1 & 2). They can either be treated on the plates they are on (In situ Differentiation Option) or transferred to new plate, slide or transwell (Cell Transfer Option).

In situ Differentiation Option: Remove media and replace with media containing fresh podocyte matrix (see Note 3)

Cell Transfer Option:

  • Coat surfaces with Laminin-521 following directions from Biolamina (see Note 4).
  • Wash cells twice with PBS.
  • Put 1 mL of Accutase in 35 mm size well (or 1 well of a 6-well plate) for 5-10 minutes in a 37° incubator.
  • Gently pipet cells up and down.
  • Add 4 mL pre-warmed Advanced RPMI/Glutamax (see Note 5) and spin for 5 minutes at 1000 rpm.
  • Remove supernatant and discard.
  • Resuspend the pellet in 1 mL of Advanced RPMI/Glutamax and count cells.
  • Transfer cells into media containing fresh podocyte matrix to the pre-coated slides/dishes/devices. We normally transfer around 45,000 cells per cm2 (see Note 6).
  • Leave the slides/dishes at room temperature for 30 minutes and then put them back into 37°C overnight.

Day 2 No media change. Leave cells alone.

Day 3 Remove media and replace with media containing fresh podocyte matrix.

Day 4 Process cells for downstream application at this day, but cells can be propagated longer by alternating days with and without media changes containing the podocyte matrix.

Critical_Steps

NOTES

  1. Embryonic Stem Cells (ESCs) or induced Pluripotent Stem Cells (iPSCs) are differentiated following the Morizane protocol as described until Day 10 (before nephron induction is initiated by CHIR treatment).
  2. NPCs can also be frozen down and used at a later time point. Cells are normally frozen down at Day 9 of the Morizane protocol. Once thawed, cells are cultured in Advanced RPMI/Glutamax containing 10 ng/ml Fgf9 for 1 day before switching over to the media containing the podocyte matrix.
  3. For 6 ml of Podocyte Matrix add the following: 1.5 ul FGF inhibitor 3.6 ul BMP4 3.6 ul BMP7 0.6 ul LPA 3 ul IWP2 0.6 ul XX inhibitor (gamma-secretase inhibitor) 0.75 ul LDN193189 in 6 ml CDM2
  4. We prefer Lamin-521, but Geltrex/Matrigel also works.
  5. Prewarm Advanced RPMI/Glutamax or CDM2 for 10 minutes before using at 37°C.
  6. 45,000 cells/cm2 results in dishes about 75% confluent on Day 2. May have to be adjusted if higher densities are required.
  7. We have used both CDM2 or Advanced RPMI/Glutamax as the basal media for the podocyte matrix. We prefer CDM2 over Advanced RPMI/Glutamax to as this contains a lipid supplement. We have seen stronger expression of the foot process markers by immunofluorescence.

Anticipated_Results

The podocytes are characterized by a robust induction of podocyte genes by qRT-PCR (e.g. WT1, MAFB, FOXC2, TCF21, FOXD1 and the structural proteins NPHS1, NPHS2 and PODXL). The establishment of stem cell-derived podocytes is corroborated by immunofluorescence, cell morphology and ultrastructural studies. The efficacy of the podocyte differentiation can be confirmed by transcriptome analysis demonstrating that the stem cell-derived podocytes most closely resemble adult podocytes found in the adult human kidney. Most importantly, the protocol does not require the addition of serum, is robust and can induce podocytes from multiple stem cell lines [Embryonic Stem Cells (ESCs) and induced pluripotent stem cells (iPSCs)] at a similar efficiency (>90% cell conversion).

References

Loh, K. M., Chen, A., Koh, P. W., Deng, T. Z., Sinha, R., Tsai, J. M., Barkal, A. A., Shen, K. Y., Jain, R., Morganti, R. M. et al. (2016). Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types. Cell 166, 451-467. Morizane, R., Lam, A. Q., Freedman, B. S., Kishi, S., Valerius, M. T. and Bonventre, J. V. (2015). Nephron organoids derived from human pluripotent stem cells model kidney development and injury. Nat Biotechnol 33, 1193-1200.

Consortium

(Re)Building a Kidney (RBK) Consortium