Differentiation of human pluripotent stem cells to multipotent neural crest cells

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...

AI Technical Summary

Benefits of technology

The present invention is about a method for creating a population of neural crest-like cells from human pluripotent stem cells without contamination from other cell types. This approach involves a single step of differentiation through WNT signaling and Activin A / Smad pathway blockade, as well as bone morphogenic protein (BMP) inhibition. The resulting cells have the potential to differentiate into peripheral neurons, mesenchymal progenitor cells, osteocytes, chondrocytes, and adipocytes. This method is efficient, feeder-layer free, and allows for the production of highly enriched neural crest-like stem cells. This invention expands opportunities for the use of neural crest cells in tissue engineering and regenerative medicine applications.

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, multistep procedures that yield relatively low yields of p75+ Hnk1+ neural crest.

These issues highlight the limitations of current approaches and consequently, severely limit their utility in scale-up applications for tissue engineering, regenerative medicine and drug screening applications.

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