Differentiation of pluripotent stem cells forming nephronoids

A technology of pluripotent stem cells, fibroblasts, applied in the field of in vitro generation of nephronoids, which can solve the problems of lack of cell types, obstacles, etc.

Active Publication Date: 2022-02-08
THE UNIV OF QUEENSLAND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the realization of human pluripotent stem cells as a cell source for clinical use and as a treatment for diseases such as kidney disease has been hampered by a lack of understanding of how to prepare the cell types necessary to generate nephrons and other structures of the kidney

Method used

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  • Differentiation of pluripotent stem cells forming nephronoids
  • Differentiation of pluripotent stem cells forming nephronoids
  • Differentiation of pluripotent stem cells forming nephronoids

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0212] Materials and methods

[0213] Non-limiting examples of sources of reagents mentioned in these methods are provided in Table 1.

[0214] The culture medium is as follows:

[0215] • Gelatin solution: 0.1% gelatin in PBS. The solution was then autoclaved.

[0216] · FDMEM: 89% DMEM High Glucose, 10% Fetal Bovine Serum, 1% GlutaMAX Supplement, 0.5% Penicillin / Streptomycin.

[0217] • KSR medium: 77.8% DMEM / F-12, 20% Knockout Serum Replacement, 1% NEAA, 1% GlutaMAX, 0.5% Penicillin / Streptomycin, 0.2% 2-mercaptoethanol (55 mM). The medium was then filtered with Stericup-GP.

[0218] • MEF-conditioned KSR medium: Add 40 mL of KSR medium to 10 million mouse embryonic fibroblasts in a T175 flask for a day. The medium was collected the next day, and 40 mL of KSR medium was added. After repeating 6 times, the collected and pooled media were filtered through Stericup-GP.

[0219]• APEL: Supplement one vial of STEMdiff APEL (100 mL) with 0.5 mL antibiotic-antimycotic.

...

Embodiment 2

[0316] Cell Culture and Differentiation

[0317] All presented experiments used the previously described wild-type human iPSC line CRL1502 (clone #C32), which uses episomal reprogramming 28 produce. Undifferentiated human iPSCs were maintained on mouse embryonic fibroblasts (MEFs) (Millipore) as a feeder layer using human ES cell (hES) medium as described previously 1 . Characterize cells and detect mycoplasma infection 28 . Human iPSCs were plated on Matrigel-coated (Millipore) dishes and cultured in MEF-conditioned hES medium (MEF-CM) until reaching 60-100% confluency. Then, the cells were grown again at 5000 cells / cm 2 Plate on Matreigel-coated dishes and culture in MEF-CM. The next day, cells reached 40–50% confluency, and cells were treated with 8 μM CHIR99021 for 2–5 days in APEL basal medium (STEMCELL Technologies) supplemented with antibiotic-antimycotics (Life Technologies) and then treated with FGF9 (200 ng mL -1) and heparin (1μg mL -1 ) for another 5-2 days...

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Abstract

Provided is a method for producing a nephrosome comprising nephrons, ureteral buds and vasculature and / or progenitor cells of these. In one embodiment, the method comprises contacting mid-mesoderm cells with fibroblast growth factor 9 and / or fibroblast growth factor 20 and / or fibroblasts under conditions that promote the formation of vascularized nephroids Cell growth factor 2, optionally one or more substances selected from the group consisting of: bone morphogenetic protein 7; heparin; Wnt agonist; retinoic acid; and RA antagonist. Another embodiment involves sequentially contacting pluripotent stem cells with a Wnt agonist and fibroblast growth factor 9 and / or fibroblast growth factor 20 and / or fibroblast growth factor 2, followed by a relatively short re-exposure to Wnt agonist to generate mesoderm cells. Nephrosomes may have end uses such as for kidney repair and regeneration, bioprinting of kidneys or their functional components, arrays of kidney cells and screening of compounds for nephrotoxicity.

Description

technical field [0001] The present invention relates to kidney development. More specifically, the present invention relates to in vitro methods of generating nephronoids comprising nephrons and / or ureteric buds and / or progenitors of these, which are at least partially vascularized and contain renal interstitial compartments. Background technique [0002] With the prevalence of end-stage renal disease rising by 8% per year globally, renal regeneration strategies are urgently needed. The kidney differentiates from the IM through the formation of the ureteric bud (UB) and the interaction between this bud and the adjacent intermediate mesoderm (IM)-derived metanephric mesenchyme (MM) Mesodermal organs. Nephrons are derived from a population of nephron progenitor cells derived from MM. Other progenitors in IM or MM are thought to contribute to renal stroma / interstitium and components of the renal vasculature including glomerular capillaries. IM itself originates from the pos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/0735
CPCC12N2501/119C12N2501/155C12N2501/16C12N2501/385C12N2501/415C12N2506/02C12N2506/45C12N5/0687C12N5/0697G01N33/5008C12N2501/91C12N5/0686C12N5/0696
Inventor 高里实梅利莎·利特尔
Owner THE UNIV OF QUEENSLAND
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