Technologies, Methods, and Products of Small Molecule Directed Tissue and Organ Regeneration from Human Pluripotent Stem Cells

Abstract

Pluripotent human embryonic stem cells (hESCs) hold great potential for restoring tissue and organ function, which has been hindered by inefficiency and instability of generating desired cell types through multi-lineage differentiation. This instant invention is based on the discovery that pluripotent hESCs maintained under defined culture conditions can be uniformly converted into a specific lineage by small molecule induction. Retinoic acid induces specification of neuroectoderm direct from the pluripotent state of hESCs and triggers progression to neuronal progenitors and neurons efficiently. Similarly, nicotinamide induces specification of cardiomesoderm direct from the pluripotent state of hESCs and triggers progression to cardiac precursors and cardiomyocytes efficiently. This technology provides a large supply of clinically-suitable human neuronal or cardiac therapeutic products for CNS or myocardium repair. This invention enables well-controlled efficient induction of pluripotent hESCs exclusively to a specific clinically-relevant lineage for tissue and organ engineering and regeneration, cell-based therapy, and drug discovery.

What is claimed as the invention is: 
     
         1 . A method of producing cells of neuronal lineage directly from pluripotent human stem cells, comprising steps of:
 (i) providing a culture of pluripotent human stem cells in a defined medium comprising bFGF, insulin, ascorbic acid, and activin-A, wherein said defined medium is free of serum, free of feeder cells, and free of feeder cell conditioned medium;   (ii) adding retinoic acid (RA) to the culture, wherein the pluripotent human stem cells are cultured in the presence of RA for a period of time sufficient to cause Nurr-1 to translocate to the nucleus and cause the pluripotent human stem cells to directly differentiate into cells of neuronal lineage comprising at least 90% of neuroectodermal cells;   (iii) further differentiating the neuroectodermal cells into a population of cells comprising at least 90% of neuronal progenitor cells; and   (iv) further differentiating the neuronal progenitor cells into a population of cells comprising at least 90% of neuronal cells.   
     
     
         2 . The method according to  claim 1 , wherein said defined medium comprises: (a) a basal medium; (b) 20-100 ng/ml bFGF; (c) 10-30 microgram/ml insulin; (d) 40-60 microgram/ml ascorbic acid; and (e) 20-100 ng/ml activin-A. 
     
     
         3 . The method according to  claim 1 , wherein at least 70% of the pluripotent human stem cells from the culture of step (i) are positive for at least 4 markers selected from the group consisting of alkaline phosphatase, Oct-4, SSEA-4, Tra-1-60, Tra-1-81, acetylated histones, Brg-1, hSNF2H, and microRNA hsa-miR-302. 
     
     
         4 . The method according to  claim 1 , wherein the pluripotent human stem cells comprise a pluripotent human embryonic stem cell line which (i) is derived from the inner cell mass or epiblast of a human blastocyst; (ii) is capable of proliferation in culture for over one year in a defined medium comprising bFGF, insulin, ascorbic acid, and activin-A, wherein said defined medium is free of serum, free of feeder cells, and free of feeder cell conditioned medium; (iii) maintains a stable karyotype in which the chromosomes are euploid through prolonged culture; and (iv) maintains the potential to differentiate to derivatives of endoderm, mesoderm, and ectoderm tissues throughout the culture. 
     
     
         5 . The method according to  claim 1 , wherein the cells of neuronal lineage comprise human neuroectodermal cells, human neural stem cells, human neuronal progenitor cells, human neural cells, human neuronal cells, human pigmented neuronal cells, human neurons, human dopaminergic neurons, human motor neurons, human ventral mesencephalon precursors, and human ventral neurons. 
     
     
         6 . The method according to  claim 1 , wherein said neuroectodermal cells are positive for at least 4 markers selected from the group consisting of Nurr-1, AP2, TrkC, beta-III-Tubulin, SSEA-1, microRNA hsa-miR-10, acetylated histones, Brg-1, and hSNF2H. 
     
     
         7 . The method according to  claim 1 , wherein said neuronal progenitor cells are positive for at least 3 markers selected from the group consisting of Nurr-1, beta-III-Tubulin, Map-2, microRNA hsa-miR-10, microRNA hsa-miR-9, acetylated histones, Brg-1, and hSNF2H. 
     
     
         8 . The method according to  claim 1 , wherein said neuronal cells are positive for at least 3 markers selected from the group consisting of Nurr-1, beta-III-Tubulin, Map-2, NeuN, 70 KDa NF, 160 KDa NF, microRNA hsa-let-7, microRNA hsa-miR-143, microRNA hsa-miR-124, and microRNA hsa-miR-210. 
     
     
         9 . The method according to  claim 1 , wherein said neuronal cells comprise dopaminergic neurons and motor neurons that are positive for at least one marker selected from the group consisting of tyrosine hydroxylase, Lmx1, Msx1, Pitx3, HB9, Lim3, and Isl1. 
     
     
         10 . The method according to  claim 1 , wherein the cells of neuronal lineage are negative for at least 4 markers selected from the group consisting of Oct-4, SSEA-4, Tra-1-60, Tra-1-81, microRNA hsa-miR-302, Nestin, Sox-2, Musashi, Pdxl, AFP, Nkx2.5, MBP, and GFAP. 
     
     
         11 . A method of neuronal cell therapy, comprising (i) producing the cells of neuronal lineage directly from pluripotent human stem cells according to the method of  claim 1 ; and (ii) administering to a patient in need of such neuronal cell therapy product the cells of neuronal lineage. 
     
     
         12 . The method according to  claim 11  wherein the patient suffers from a neurological disorder comprising neurodegenerative diseases, spinal cord injury, motor neuron disease, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, spinal muscular atrophy, brain injury, stroke, and macular degeneration. 
     
     
         13 . The method according to  claim 11 , wherein the pluripotent human stem cells comprise a pluripotent human embryonic stem cell line. 
     
     
         14 . A method of nerve system drug discovery, comprising (i) producing the cells of neuronal lineage directly from pluripotent human stem cells according to the method of  claim 1 , and (ii) a high throughput method to screen a compound for an effect on such product of neuronal lineage cells. 
     
     
         15 . The method according to  claim 14 , wherein the pluripotent human stem cells comprise a pluripotent human embryonic stem cell line. 
     
     
         16 . A method of nerve system tissue engineering, comprising (i) producing the cells of neuronal lineage directly from pluripotent human stem cells according to the method of  claim 1 , and (ii) adding such product of neuronal lineage cells to a structural template that comprises extracellular matrix, biomaterial scaffold, whole-heart scaffold, tissue scaffold, organ scaffold, and synthetic or purified matrix proteins. 
     
     
         17 . The method according to  claim 16 , wherein the pluripotent human stem cells comprise a pluripotent human embryonic stem cell line.