US-12618044-B2 - Specification of functional cranial placode derivatives from human pluripotent stem cells

US12618044B2US 12618044 B2US12618044 B2US 12618044B2US-12618044-B2

Abstract

Cranial placodes are embryonic structures essential for sensory and endocrine organ development. The efficient derivation of cranial placodes from human pluripotent stem cells is disclosed where the timed removal of the BMP inhibitor Noggin, a component of the dual-SMAD inhibition strategy of neural induction, triggers placode induction at the expense of CNS fates. Further fate specification at the pre-placode stage enables the selective generation of placode-derived trigeminal ganglia capable of in vivo engraftment, mature lens fibers and anterior pituitary hormone-producing cells that upon transplantation produce hormones including, but not limited to, human growth hormone and adrenocortiocotropic hormone in vivo. Alternatively, anterior pituitary hormone-producing cells are generated in cell culture systems in vitro.

Inventors

Stuart Chambers
Lorenz Studer
Zehra Dincer
Bastian Zimmer

Assignees

MEMORIAL SLOAN-KETTERING CANCER CENTER

Dates

Publication Date: 20260505
Application Date: 20221221

Claims (8)

1 . A method for inducing differentiation of cells, comprising: a) contacting a plurality of cells with an inhibitor of Small Mothers Against Decapentaplegic protein signaling (“SMAD inhibitor”) and a bone morphogenetic protein (“BMP”) in amounts effective to induce detectable expression of SIX1 and PAX6 in the plurality of cells; b) contacting the cells expressing detectable levels of SIX1 and PAX6 with an activator of Wnt signaling in an amount effective to obtain differentiated cells expressing detectable levels of SIX1 and PAX3; and c) culturing the cells expressing SIX1 and PAX3 under conditions favoring differentiation of cells expressing SIX1 and PAX3 into trigeminal neurons.
2 . The method of claim 1 , wherein the method comprises contacting the cells with the SMAD inhibitor for up to about 11 days, and wherein the cells are contacted with the BMP for up to about 3 days.
3 . The method of claim 1 , wherein the cells expressing SIX1 and PAX3 are trigeminal placode cells.
4 . The method of claim 3 , wherein the cells expressing SIX1 and PAX3 further express a detectable level of GD2, CD57, or a combination thereof.
5 . The method of claim 4 , further comprising isolating the cells expressing a detectable level of GD2, CD57, or a combination thereof from the plurality of cells.
6 . The method of claim 1 , further comprising contacting the cells with a compound selected from the group consisting of BRL-54443, parthenolide, phenantroline, and combinations thereof.
7 . The method of claim 1 , wherein the cells expressing SIX1 and PAX6 further express a detectable level of TFAP2A.
8 . The method of claim 1 , wherein the conditions favoring differentiation of cells expressing SIX1 and PAX3 into trigeminal neurons comprise exposing the cells to at least one of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and DAPT.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 16/373,026 filed Apr. 2, 2019, which is a continuation of U.S. patent application Ser. No. 15/159,351 filed May 19, 2016, now U.S. Pat. No. 10,273,452, which is a continuation of International Patent Application No. PCT/US2014/066952, filed Nov. 21, 2014, which claims priority to U.S. Provisional Application No. 61/907,302, filed Nov. 21, 2013, priority to each of which is claimed, and the contents of each of which are incorporated by reference in their entireties herein. GRANT INFORMATION This invention was made with government support under grant number NS072381 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION The present invention relates generally to the field of cell biology of stem cells, more specifically the directed differentiation of pluripotent or multipotent stem cells, including human embryonic stem cells (hESC), somatic stem cells, and induced human pluripotent stem cells (hiPSC) using novel culture conditions. In particular, cranial placodes are derived from human pluripotent stem cells by a dual-SMAD inhibition strategy of neural induction coupled with further fate specification at the pre-placode stage. The method generates placode-derived trigeminal ganglia, mature lens fibers and anterior pituitary hormone-producing cells. Applications of these cells include, but are not limited to, human cell-based therapies in sensory and endocrine disease. SEQUENCE LISTING The specification further incorporates by reference the Sequence Listing submitted herewith via EFS on Dec. 21, 2022. Pursuant to 37 C.F.R. § 1.52(e)(5), the Sequence Listing xml file, identified as 0727341429_SL, is 3,250 bytes and was created on Dec. 20, 2022. The Sequence Listing, electronically filed herewith, does not extend beyond the scope of the specification and thus does not contain new matter. BACKGROUND OF THE INVENTION The differentiation capacity of embryonic and somatic stem cells have opened possibilities for cell replacement therapies for genetic, malignant, and degenerative diseases. Neurodegenerative disorders, conditions, and diseases, and their treatment by cell-based therapies represent a promising means of preventing, reducing or eliminating the symptoms. Such disorders include Huntington's disease, Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis. They also provide a source of cells for screening for critical small molecules (i) that could be useful in for treatment of disease; or (ii) for determining the cell fate of neural tissue. Further, these cells were studied in order to characterize key genes, mRNA transcripts, and proteins relevant in normal or pathological lineages. Neural development is dictated in time and space by a complex set of signals that instruct neural precursor identity. While significant progress was made in animal models, human neural development remains much less understood. Previous studies reported directed differentiation of mouse (Wichterle et al., 2002; Barberi et al., 2003; Watanabe et al., 2005) and human (Perrier et al., 2004; Li et al., 2008; Eiraku et al., 2008) ESCs into specific neuron types in response to patterning factors defining anterior/posterior (A/P) and dorso-/ventral (D/V) CNS identity. These studies demonstrate evolutionary conservation of signaling systems that specify the major CNS regions. In mammals, sonic hedgehog (SHH) is a ventralizing factor acting in a dose-dependent manner to specify the various ventral cell types including cells expressing floor plate (FP) in primary neural explants (Briscoe and Ericson, 1999) and in mouse ES cells (Mizuseki et al., 2003). While application of SHH to hESC-derived neural cells was shown to induce various ventral neuron types, the derivation of floor plate (FP) tissue itself was not reported. As FP is one signaling center for inducing differentiation pathways and subsequent committed cell linage, the ability to produce FP from human ES cells would be a major step forward in furthering studies of early human neural development. Furthermore, little is known about FP development in humans, due to lack of accessibility to tissue. In animals, the FP is a major site of SHH production and several human developmental disorders are related to alterations in midline SHH signaling (Mullor et al., 2002) including certain forms of holoprosencephaly and microphthalmia, skeletal disorders including various cleft plate syndromes, and tumor conditions such as Gorlin's syndrome; a rare genetic disorder caused by a mutation in the SHH receptor Patched 1. However it is not known whether similar alterations in midline SHH signaling would induce these diseases in humans. Therefore there is a critical need for inducing human floor plate tissue from human embryonic stem cells (hESCs) for providing a source of human floor plate cells. These human floor pla