US2022168359A1PendingUtilityA1

Methods and compositions for producing hepatocyte-like cells

73
Assignee: UNIV LELAND STANFORD JUNIORPriority: Sep 19, 2013Filed: Feb 15, 2022Published: Jun 2, 2022
Est. expirySep 19, 2033(~7.1 yrs left)· nominal 20-yr term from priority
Inventors:Gary PeltzDan Xu
C12N 5/067C12N 2501/119C12N 2501/16C12N 2501/415C12N 2501/606C12N 2501/39C12N 2501/155C12N 2501/12C12N 2506/45C12N 2501/602C12N 2506/1384C12N 2533/90C12N 2501/604C12N 5/0696C12N 2500/62A61P 9/10C12N 2510/00A61P 3/10C12N 2513/00C12N 2501/603A61K 35/407C12N 2501/237
73
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Claims

Abstract

Methods are provided for producing a population of hepatocyte-like cells (iHeps) from a population of adipocyte-derived stem cells (ASCs). Aspects of the methods include placing a population of ASCs into a three dimensional culture (e.g., hanging drop suspension culture, high density culture, spinner flask culture, microcarrier culture, etc.), and contacting the cells with a first and second culture medium. Also provided are methods of treating an individual, which include producing a population of iHeps from a population of ASCs, and administering an effective number of iHeps into the individual. Kits for practicing the methods are also described herein.

Claims

exact text as granted — not AI-modified
That which is claimed is: 
     
         1 . A method of producing a population of hepatocyte-like cells from a population of adipocyte-derived stem cells (ASCs), the method comprising:
 (a) placing a population of ASCs into a three dimensional culture for a period of time sufficient to produce an ASC-derived cellular aggregate;   (b) contacting cells of the ASC-derived cellular aggregate with a first culture medium comprising Activin A and a fibroblast growth factor (FGF) to produce a precursor cell population; and   (c) contacting cells of the precursor cell population with a second culture medium comprising hepatocyte growth factor (HGF) to produce an induced cell population that comprises induced hepatocyte-like cells (iHeps),   wherein the total time elapsed from beginning step (a) to the production of an induced cell population in step (c) is less than 13 days.   
     
     
         2 . The method of  claim 1 , wherein the three dimensional culture is a spinner flask culture. 
     
     
         3 . The method of  claim 1 , wherein the three dimensional culture is a microcarrier culture. 
     
     
         4 . The method of  claim 1 , wherein the three dimensional culture is a hanging drop suspension culture. 
     
     
         5 . The method of  claim 1 , wherein the total time elapsed from beginning step (a) to the production of an induced cell population in step (c) is 10 days or less. 
     
     
         6 . The method of  claim 1 , wherein the period of time sufficient to produce an ASC-derived cellular aggregate is 3 days or less. 
     
     
         7 . The method of  claim 1 , wherein the cells of the ASC-derived cellular aggregate are contacted with the first culture medium for 7 days or less. 
     
     
         8 . The method of  claim 1 , wherein the cells of the ASC-derived cellular aggregate are contacted with the first culture medium for 7 days or less. 
     
     
         9 . The method of  claim 1 , wherein the FGF is FGF 4. 
     
     
         10 . The method of  claim 1 , wherein the first culture medium further comprises a Wnt signaling agonist. 
     
     
         11 . The method of  claim 3 , wherein the Wnt signaling agonist is Wnt3a. 
     
     
         12 . The method of  claim 1 , wherein the second culture medium further comprises a compound selected from the group consisting of: oncostatinM (OSM), dexamethasone (Dex), Dimethyl sulfoxide (DMSO), and a combination thereof. 
     
     
         13 . The method of  claim 1 , wherein the second culture medium further comprises oncostatinM (OSM), dexamethasone (Dex), and Dimethyl sulfoxide (DMSO). 
     
     
         14 . The method of  claim 1 , further comprising determining the percentage of cells of the induced cell population that are iHeps. 
     
     
         15 . The method of  claim 14 , wherein determining the percentage of cells comprises:
 contacting cells of the induced cell population with a specific binding agent for a hepatocyte marker molecule; and   determining the percentage of cells positive for expression of the hepatocyte marker molecule,   wherein cells positive for expression of the hepatocyte marker molecule are iHeps.   
     
     
         16 . The method of  claim 14 , wherein 11% or more of the cells of the induced cell population are iHeps. 
     
     
         17 . The method of  claim 16 , wherein 25% or more of the cells of the induced cell population are iHeps. 
     
     
         18 . The method of  claim 1 , further comprising enriching the induced cell population for iHeps. 
     
     
         19 . The method of  claim 18 , wherein the enriching comprises fluorescent activated cell sorting (FACS). 
     
     
         20 . A method of treating an individual with reduced liver function, the method comprising:
 (a) producing a population of hepatocyte-like cells from a population of adipose-derived stem cells (ASCs) by a method comprising:   (i) placing a population of ASCs into a three dimensional culture for a period of time sufficient to produce an ASC-derived cellular aggregate,   (ii) contacting cells of the ASC-derived cellular aggregate with a first culture medium comprising Activin A and a fibroblast growth factor (FGF) to produce a precursor cell population, and   (iii) contacting cells of the precursor cell population with a second culture medium comprising hepatocyte growth factor (HGF) to produce an induced cell population that comprises induced hepatocyte-like cells (iHeps),   wherein the total time elapsed from beginning step (i) to the production of an induced cell population in step (iii) is less than 13 days; and   (b) administering an effective number of iHeps into the individual to improve liver function.   
     
     
         21 . The method of  claim 20 , wherein the three dimensional culture is a spinner flask culture. 
     
     
         22 . The method of  claim 20 , wherein the three dimensional culture is a microcarrier culture. 
     
     
         23 . The method of  claim 20 , wherein the three dimensional culture is a hanging drop suspension culture. 
     
     
         24 . The method of  claim 20 , wherein the total time elapsed from beginning step (a) to the production of an induced cell population in step (c) is 10 days or less. 
     
     
         25 . The method of  claim 20 , wherein the period of time sufficient to produce an ASC-derived cellular aggregate is 3 days or less. 
     
     
         26 . The method of  claim 20 , wherein the cells of the ASC-derived cellular aggregate are contacted with the first culture medium for 7 days or less. 
     
     
         27 . The method of  claim 20 , wherein the cells of the ASC-derived cellular aggregate are contacted with the first culture medium for 7 days or less. 
     
     
         28 . The method of  claim 20 , wherein the FGF is FGF 4. 
     
     
         29 . The method of  claim 20 , wherein the first culture medium further comprises a Wnt signaling agonist. 
     
     
         30 . The method of  claim 29 , wherein the Wnt signaling agonist is Wnt3a. 
     
     
         31 . The method of  claim 20 , wherein the second culture medium further comprises a compound selected from the group consisting of: oncostatinM (OSM), dexamethasone (Dex), Dimethyl sulfoxide (DMSO), and a combination thereof. 
     
     
         32 . The method of  claim 20 , wherein the total time elapsed from beginning step (i) to producing an induced cell population that comprises induced hepatocyte-like cells (iHeps) in step (iii) is less than 13 days. 
     
     
         33 . The method of  claim 20 , further comprising determining the percentage of cells of the induced cell population that are iHeps. 
     
     
         34 . The method of  claim 33 , wherein determining the percentage of cells comprises:
 contacting cells of the induced cell population with a specific binding agent for a hepatocyte marker molecule; and   determining the percentage of cells positive for expression of the hepatocyte marker molecule,   wherein cells positive for expression of the hepatocyte marker molecule are iHeps.   
     
     
         35 . The method of  claim 20 , wherein 15% or more of the cells of the induced cell population are iHeps. 
     
     
         36 . The method of  claim 35 , wherein 25% or more of the cells of the induced cell population are iHeps. 
     
     
         37 . The method of  claim 20 , further comprising, prior to step (b), enriching the induced cell population for iHeps. 
     
     
         38 . The method of  claim 37 , wherein enriching comprises fluorescent activated cell sorting (FACS). 
     
     
         39 . The method of  claim 20 , wherein at least 1×10 4  iHeps are administered. 
     
     
         40 . The method of  claim 20 , wherein the iHeps are transplanted into the liver. 
     
     
         41 . The method of  claim 40 , wherein the iHeps are transplanted into the liver using ultrasound guided injection. 
     
     
         42 . The method of  claim 20 , wherein the individual is a mammal. 
     
     
         43 . The method of  claim 42 , wherein the mammal is a human. 
     
     
         44 . The method of  claim 20 , wherein the ASCs are ASCs isolated from the individual. 
     
     
         45 . The method of  claim 44 , further comprising, prior to step (a), isolating the ASCs from the individual.

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