Neural progenitor cell differentiation

Patent metadata

Field	Value
Publication number	US-2016017285-A1
Application number	US-201414777335-A
Country	US
Kind code	A1
Filing date	Mar 17, 2014
Priority date	Mar 15, 2013
Publication date	Jan 21, 2016
Grant date	—

Abstract

Differentiation and stability of neural stem cells can be enhanced by in vitro or in vivo culturing with one or more extracellular matrix (ECM) compositions, such as collagen I, IV, laminin and/or a heparan sulfate proteoglycan. In one aspect of the invention, adult mammalian enteric neuronal progenitor cells can be induced to differentiate on various substrates derived from components or combinations of neural ECM compositions. Collagen I and IV supported neuronal differentiation and extensive glial differentiation individually and in combination. Addition of laminin or heparan sulfate to collagen substrates unexpectedly improved neuronal differentiation, increasing neuron number, branching of neuronal processes, and initiation of neuronal network formation. In another aspect, neuronal subtype differentiation was affected by varying ECM compositions in hydrogels overlaid on intestinal smooth muscle sheets. The matrix compositions of the present invention can be used to tissue engineer transplantable innervated GI smooth muscle constructs to remedy aganglionic disorders.

First claim

1 . A method of biasing neural stem cell differentiation comprising:
obtaining a population of smooth muscle cells; culturing the smooth muscle cells to form a uniaxially-aligned smooth muscle sheet; obtaining a population of neural stem cells; culturing the neural stem cells in a hydrogel, wherein the hydrogel is applied to the uniaxially-aligned smooth muscle sheet; and exposing the neural stem cells to at least one extracellular matrix (ECM) component, wherein the ECM component biases differentiation of the neural stem cells into differentiated neural stem cells that are enriched for a neuronal subtype.

2 . The method of claim 1 , wherein the neuronal subtype are cholinergic neurons.

3 . The method of claim 2 , wherein the hydrogel comprises collagen I.

4 . The method of claim 2 , wherein the hydrogel comprises at least about 800 μg/ml collagen I.

5 . The method of claim 2 , wherein the hydrogel comprises between about 800 μg/ml and about 1600 μg/ml collagen I.

6 . The method of claim 1 , wherein the neuronal subtype are nitrergic neurons.

7 . The method of claim 6 , wherein the hydrogel comprises collagen IV and is substantially free of laminin.

8 . The method of claim 6 , wherein the hydrogel comprises at least about 200 μg/ml collagen IV and is substantially free of laminin.

9 . The method of claim 1 , wherein the neuronal subtype are peptidergic neurons.

10 . The method of claim 9 , wherein the hydrogel comprises collagen I, collagen IV, and laminin.

11 . The method of claim 9 , wherein the hydrogel comprises at least about 800 μg/ml collagen I, at least about 200 μg/ml collagen IV, and at least about 5 μg/ml laminin.

12 . The method of claim 1 , further comprising isolating the differentiated neural stem cells and administering the differentiated neural stem cells to a patient.

13 . The method of claim 12 , wherein the administering comprises injecting, into the patient, the differentiated stem cells in the hydrogel.

14 . The method of claim 1 , wherein the differentiated neural stem cells innervate the uniaxially-aligned smooth muscle sheet to form an innervated smooth muscle sheet.

15 . The method of claim 14 , further comprising implanting the innervated smooth muscle sheet into a patient.

16 . A method of biasing neural stem cell differentiation comprising:
obtaining a population of neural stem cells; obtaining a population of smooth muscle cells; culturing the neural stem cells in the presence of the smooth muscle cells, wherein the neural stem cells adhere on a substrate with a substrate coating comprising at least one extracellular matrix (ECM) component, wherein the ECM component biases differentiation of the neural stem cells into differentiated neural stem cells that are enriched for neurons.

17 . The method of claim 16 , wherein the substrate coating comprises at least one of laminin, collagen I, and collagen IV.

18 . The method of claim 16 , wherein the substrate coating comprises laminin, and at least one of collagen I and collagen IV.

19 . The method of claim 16 , wherein the substrate coating comprises collagen I and collagen IV, and at least one of laminin and heparan sulfate.

20 . The method of claim 16 , further comprising isolating the differentiated neural stem cells and administering the differentiated neural stem cells to a patient.

21 . The method of claim 20 , wherein the administering comprises injecting the differentiated neural stem cells into the patient.

22 . A method of biasing neural stem cell differentiation comprising:
obtaining a population of neural stem cells; obtaining a population of smooth muscle cells; culturing the neural stem cells in the presence of the smooth muscle cells, wherein the neural stem cells adhere on a substrate with a substrate coating comprising at least one extracellular matrix (ECM) component, wherein the ECM component biases differentiation of the neural stem cells into differentiated neural stem cells that are enriched for glial cells.

23 . The method of claim 22 , wherein the substrate coating comprises at least collagen I and collagen IV, and substantially free of at least one of laminin and heparan sulfate.

24 . The method of claim 22 , wherein the substrate coating comprises at least 5 μg/cm 2 collagen I and at least 5 μg/cm 2 collagen IV, and substantially free of at least one of laminin and heparan sulfate.

25 . The method of claim 22 , further comprising isolating the differentiated neural stem cells and administering the differentiated neural stem cells to a patient.

26 . The method of claim 25 , wherein the administering comprises injecting the differentiated neural stem cells into the patient.

Assignees

• Univ Wake Forest Health Sciences

Inventors

• Bitar Khalil

Classifications

• A61K35/30

  Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue · CPC title

• C12N2533/90

  Substrates of biological origin, e.g. extracellular matrix, decellularised tissue · CPC title

• C12N5/0622

  Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells · CPC title

• A61K35/34

  Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes (vascular smooth muscle A61K35/44) · CPC title

• C12N5/0619 Primary

  Neurons · CPC title