US2015203814A1PendingUtilityA1

Methods of isolating and culturing stem cells

58
Assignee: TISSUETECH INCPriority: Mar 2, 2012Filed: Dec 22, 2014Published: Jul 23, 2015
Est. expiryMar 2, 2032(~5.6 yrs left)· nominal 20-yr term from priority
Inventors:Scheffer TsengSzu-Yu ChenSuzhen Zhang
C12N 2533/80C12N 2501/235C12N 2500/34C12N 2533/90C12N 5/0667C12N 2501/115C12N 5/0665C12N 5/0607C12N 2501/998C12N 2501/40
58
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Claims

Abstract

Provided herein are methods of isolating and expanding a plurality of multipotent stem cells. Also described are methods of expanding stem cells on a substrate comprising an HC-HA complex. Also described are isolated and expanded stem cells produced by the methods and uses thereof, including stem cell therapy, as niche cells for supporting other types of stem cells, or as bioreactors for the production of HC-HA complexes. Also described are uses of HC-HA complexes as a carrier for stem cells.

Claims

exact text as granted — not AI-modified
1 . A method for expanding a stem cell population comprising:
 expanding one or more isolated stem cells of  claim 1  in a culture comprising a two-dimensional substrate, thereby forming a plurality of expanding stem cells.   
     
     
         2 . The method of  claim 1 , wherein the two-dimensional substrate comprises an HC-HA complex. 
     
     
         3 . The method of  claim 2 , wherein the HC-HA complex is immobilized. 
     
     
         4 . The method of  claim 2 , wherein the HC-HA complex is a native HC-HA complex or is a reconstituted HC-HA complex. 
     
     
         5 . The method of  claim 2 , wherein the native HC-HA complex is an amniotic membrane HC-HA complex. 
     
     
         6 . The method of  claim 2 , wherein the HC-HA complex comprises TSG-6. 
     
     
         7 . The method of  claim 2 , wherein the HC-HA complex comprises PTX3. 
     
     
         8 . The method of  claim 2 , wherein the HC-HA complex comprises a small leucine-rich proteoglycan. 
     
     
         9 . The method of  claim 8 , wherein the small leucine-rich proteoglycan is selected from among decorin, biglycan, fibromodulin, lumican, PRELP (proline arginine rich end leucine-rich protein), keratocan, osteoadherin, epipycan, and osteoglycin. 
     
     
         10 . The method of  claim 2 , wherein the HC-HA complex comprises TSG-6, PTX3, and a SLRP. 
     
     
         11 . The method of  claim 1 , wherein the culture comprises bFGF or LIF. 
     
     
         12 . A method for expanding a stem cell population comprising:
 expanding one or more stem cells in a culture comprising a two-dimensional substrate comprising an HC-HA complex, thereby forming a plurality of expanding stem cells.   
     
     
         13 . The method of  claim 12 , wherein at least one of the expanding stem cells does not pass the Hayflick limit. 
     
     
         14 . The method of  claim 12 , wherein the HC-HA complex is immobilized. 
     
     
         15 . The method of  claim 12 , wherein the HC-HA complex is a native HC-HA complex or is a reconstituted HC-HA complex. 
     
     
         16 . The method of  claim 12 , wherein the native HC-HA complex is an amniotic membrane HC-HA complex. 
     
     
         17 . The method of claim  4816 , further comprising purifying the native HC-HA complex by performing ultracentrifugation on an amniotic membrane extract or an umbilical cord extract. 
     
     
         18 . The method of  claim 17 , further comprising performing two, three or four rounds of ultracentifugation. 
     
     
         19 . The method of  claim 17 , further comprising performing four rounds of ultracentifugation. 
     
     
         20 . The method of  claim 12 , wherein the HC-HA complex comprises TSG-6. 
     
     
         21 . The method of  claim 12 , wherein the HC-HA complex comprises PTX3. 
     
     
         22 . The method of  claim 12 , wherein the HC-HA complex comprises a small leucine-rich proteoglycan. 
     
     
         23 . The method of  claim 22 , wherein the small leucine-rich proteoglycan is selected from among decorin, biglycan, fibromodulin, lumican, PRELP (proline arginine rich end leucine-rich protein), keratocan, osteoadherin, epipycan, and osteoglycin. 
     
     
         24 . The method of  claim 12 , wherein the HC-HA complex comprises TSG-6, PTX3, and a SLRP. 
     
     
         25 . The method of  claim 12 , wherein the stem cell is an embryonic stem cell, an adult stem cell, a fetal stem cell, or an induced progenitor cell. 
     
     
         26 . The method of  claim 12 , wherein the stem cell is a limbal stromal niche cell, an umbilical cord stem cell, an amniotic membrane stem cell or an adipose stem cell. 
     
     
         27 . The method of  claim 12 , wherein the stem cell is a mesenchymal stem cell. 
     
     
         28 . The method of  claim 12 , wherein the stem cell is a stem cell derived from an adult differentiated cell. 
     
     
         29 . The method of  claim 12 , wherein the stem cell is a stem cell derived from a fibroblast. 
     
     
         30 . The method of  claim 29 , wherein the stem cell is derived from a conjunctivochalasis fibroblast. 
     
     
         31 . A method for expanding a stem cell comprising:
 (a) separating a plurality of cells of a tissue sample from components of an extracellular matrix in the tissue sample, to form a mixed cell population;   (b) culturing the mixed cell population in a first culture comprising supplemented hormonal epithelial medium (SHEM) on a plastic tissue culture dish, thereby producing a population of non-adherent cells;   (c) isolating the population of non-adherent cells; and   (d) expanding at least one cell of the population of non-adherent cells in a second culture comprising a two-dimensional substrate, thereby forming a plurality of expanding stem cells.   
     
     
         32 . The method of  claim 31 , wherein at least one of the expanding stem cells does not pass the Hayflick limit. 
     
     
         33 . The method of  claim 32 , wherein the tissue is an amniotic membrane, an umbilical cord, a limbal tissue or an adipose tissue. 
     
     
         34 . The method of  claim 32 , wherein the stem cell is a limbal stromal niche cell, an umbilical cord stem cell, an amniotic membrane stem cell or an adipose stem cell. 
     
     
         35 . The method of  claim 32 , wherein the stem cell is a mesenchymal stem cell. 
     
     
         36 . The method of  claim 32 , wherein the two-dimensional substrate comprises Matrigel, laminin, fibronectin, collagen or entactin. 
     
     
         37 . The method of  claim 32 , wherein the two-dimensional substrate comprises an HC-HA complex. 
     
     
         38 . The method of  claim 37 , wherein the HC-HA complex is a native HC-HA complex or is a reconstituted HC-HA complex. 
     
     
         39 . The method of  claim 38 , wherein the native HC-HA complex is an amniotic membrane HC-HA complex. 
     
     
         40 . The method of  claim 38 , further comprising purifying the native HC-HA complex by performing ultracentrifugation on an amniotic membrane extract or an umbilical cord extract. 
     
     
         41 . The method of  claim 40 , further comprising performing two, three or four rounds of ultracentifugation. 
     
     
         42 . The method of  claim 40 , further comprising performing four rounds of ultracentifugation. 
     
     
         43 . The method of  claim 37 , wherein the HC-HA complex comprises TSG-6. 
     
     
         44 . The method of  claim 37 , wherein the HC-HA complex comprises PTX3. 
     
     
         45 . The method of  claim 37 , wherein the HC-HA complex comprises a small leucine-rich proteoglycan. 
     
     
         46 . The method of  claim 45 , wherein the small leucine-rich proteoglycan is selected from among decorin, biglycan, fibromodulin, lumican, PRELP (proline arginine rich end leucine-rich protein), keratocan, osteoadherin, epipycan, and osteoglycin. 
     
     
         47 . The method of  claim 37 , wherein the HC-HA complex comprises TSG-6, PTX3, and a SLRP. 
     
     
         48 . A composition comprising a plurality of expanded stem cells of  claim 1  and a pharmaceutically acceptable excipient. 
     
     
         49 . A composition comprising:
 (a) a stem cell;   (b) an HC-HA complex; and   (c) a tissue culture plate.   
     
     
         50 . The composition of  claim 49 , wherein the HC-HA complex is immobilized to the tissue culture plate. 
     
     
         51 . The composition of  claim 49 , wherein the stem cell is an isolated or expanded stem cell of  claim 1 . 
     
     
         52 . The composition of  claim 49 , wherein the stem cell is an embryonic stem cell, an adult stem cell, a fetal stem cell, or an induced progenitor cell. 
     
     
         53 . The composition of  claim 49 , wherein the stem cell is a limbal stromal niche cell, an umbilical cord stem cell, an amniotic membrane stem cell or an adipose stem cell. 
     
     
         54 . The composition of  claim 49 , wherein the stem cell is a mesenchymal stem cell. 
     
     
         55 . The composition of  claim 49 , wherein the stem cell is an induced pluripotent stem cell derived from an adult differentiated cell. 
     
     
         56 . The composition of  claim 49 , wherein the stem cell is an induced pluripotent stem cell derived from a fibroblast. 
     
     
         57 . The composition of  claim 49 , wherein the stem cell is an induced pluripotent stem cell derived from a conjunctivochalasis fibroblast. 
     
     
         58 . The composition of  claim 49 , wherein the HC-HA complex is a native HC-HA complex or is a reconstituted HC-HA complex.

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