Neural progenitor cell differentiation

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.

1 - 15 . (canceled) 
     
     
         16 . A method of preparing an innervated smooth muscle construct, comprising the steps of:
 obtaining a population of smooth muscle cells,   culturing the population of smooth muscle cells in a first extracellular matrix (ECM) substrate,   obtaining a population of neural progenitor cells,   culturing the population of neural progenitor cells in a second ECM substrate,   seeding a multi-layer matrix on a mold, comprising at least one layer of smooth muscle cells in said first ECM substrate and at least one layer of neural progenitor cells in in said second ECM substrate in contact with one another, wherein the matrix is seeded in a substantially circular shape, and   contacting the multi-layer gel matrix with a differentiation medium that promoted neuronal differentiation, thereby forming the innervated smooth muscle construct.   
     
     
         17 . The method of  claim 16  wherein the population of neural progenitor cells is obtained from embryonic or adult central nervous system tissue, neural tube tissue or enteric nervous system (ENS) tissue. 
     
     
         18 . The method of  claim 17  wherein the population of neural progenitor cells is obtained from enteric neurospheres. 
     
     
         19 . The method of  claim 16 , wherein the first ECM substrate comprises collagen. 
     
     
         20 . The method of  claim 16 , wherein the second ECM substrate comprises collagen and at least one of laminin or heparin sulfate. 
     
     
         21 . The method of  claim 20  wherein the second ECM substrate further comprises a hydrogel. 
     
     
         22 . The method of  claim 20  wherein the second ECM substrate comprises at least 800 μg/ml of collagen type I. 
     
     
         23 . The method of  claim 20 , wherein the second ECM substrate comprises between about 800 μg/ml and about 1600 μg/ml collagen I. (New) The method of  claim 20  wherein the second ECM substrate further comprises at least 200 .μg/ml of collagen type IV. 
     
     
         25 . The method of  claim 20  wherein the second ECM substrate further comprises at least 5 μg/ml of laminin. 
     
     
         26 . The method of  claim 20  wherein the second ECM substrate is substantially free of laminin. 
     
     
         27 . The method of  claim 20  wherein the second ECM substrate is substantially free of heparan sulfate. 
     
     
         28 . The method of  claim 16  wherein the construct is a tubular construct. 
     
     
         29 . The method of  claim 16  wherein the method further comprises administering the construct to a patient. 
     
     
         30 . The method of  claim 29 , wherein the step of administering the construct further comprises implanting the construct into the patient.