Differentiation of human pluripotent stem cells to multipotent neural crest cells

Inactive Publication Date: 2013-10-24
UNIV OF GEORGIA RES FOUND INC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0007]The present invention relates to a method for producing a population of p75+ Hnk1+ Ap2+ multipotent neural crest-like cells from human pluriopotent cells, preferably human pluriopotent stem cells or human induced pluripotent cells in a single step without appreciable contamination with Pax6+ neural progenitor cells in the population of neural crest-like stem cells produced. This approach, which involves differentiation of pluripotent cells through WNT signali

Problems solved by technology

Alternative methods for generating neural crest cells from pluripotent cells have been described, but these utilize co-culture on feeder layers (16, 17), are relatively inefficient and also require cell sorting to generate highly enriched populations.
These methods all involve complex, mul

Method used

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  • Differentiation of human pluripotent stem cells to multipotent neural crest cells
  • Differentiation of human pluripotent stem cells to multipotent neural crest cells
  • Differentiation of human pluripotent stem cells to multipotent neural crest cells

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Materials and Methods

Stem Cell Culture.

[0080]WA09 (WiCell), RUES1, RUES2 (Dr. A. Brivanlou, Rockefeller University) hESCs and the hiPSC lines Fib2-iPS4 and Fib2-iPS5 (Dr. George Daley, Children's Hospital Boston) were cultured on Geltrex (Invitrogen)-coated plates in chemically defined media containing Heregulin 13 (10 ng / ml), Activin A (10 ng / ml), LR-Igf (200 ng / ml) and Fgf2 (8 ng / ml) as described previously (25).

Neuroprogenitor Cell, Neural Crest and Mesenchymal Cell Differentiation.

[0081]NPC differentiation was performed as described (18). Briefly, cells were plated on Geltrex-coated plates in defined media without Activin A, supplemented with 20 μM SB 431542 (Tocris) and 500 ng / ml Noggin (R&D systems) for 11 days with or without 2 μM of (2′Z,3′E)-6-Bromoindirubin-3′-oxime (BIO) (GSK3 Inhibitor IX, Calbiochem), or 15 ng / ml Dickkopf (R&D Systems). For direct neural crest differentiation, cells were plated at a density of 1×105 cells / cm2 in defined media lacking Activin A, suppleme...

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Abstract

The present invention relates to the differentiation of human pluripotent cells, including human pluripotent stems cells to produce a self-renewing multipotent neural crest cell population in a single step method without the requirement of isolation of intermediate cells and without appreciable contamination (in certain preferred instances, virtually none) with Pax6+ neural progenitor cells in the population of p75+ Hnk1+ Ap2+ multipotent neural crest-like cells. The multipotent neural crest cell population obtained can be clonally amplified and maintained for >25 passages (>100 days) while retaining the capacity to differentiate into peripheral neurons, smooth muscle cells and mesenchymal precursor cells.

Description

RELATED APPLICATIONS AND GRANT SUPPORT[0001]The present application claims priority from provisional application Nos. 61 / 428,998, filed 31 Dec. 2010; 61 / 429,344, filed 3 Jan. 2011; 61 / 429,992, filed 5 Jan. 2011 and 61 / 548,045, filed 17 Oct. 2011, each of identical title to the present application, and each of said applications being incorporated by reference in its entirety herein.[0002]This invention was made with government support under grant number HD049647 awarded by the National Institute of Child Health and grant number GM075334 awarded by the National Institute for General Medical Sciences. The government has certain rights in the invention.FIELD OF THE INVENTION[0003]The present invention relates to the differentiation of human pluripotent cells, including human pluripotent stems cells (including hiPSCs) to produce a self-renewing multipotent neural crest cell population in a single step method preferably without the requirement of isolating intermediate cells and without a...

Claims

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Application Information

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IPC IPC(8): C12N5/0797
CPCC12N5/0623C12N2501/155C12N2501/16C12N2501/415C12N2501/998C12N2506/02C12N2506/45
Inventor DALTON, STEPHENMENENDEZ, LAURA M.
Owner UNIV OF GEORGIA RES FOUND INC
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