Stem cell-derived hepatocytes in co-culture and uses thereof

• US 10,717,966 B2
• Filed: 10/11/2018
• Issued: 07/21/2020
• Est. Priority Date: 01/14/2014
• Status: Active Grant

• Current Assignee: Colorado State University Research Foundation
• Original Assignee: Colorado State University Research Foundation
• Inventors: Khetani, Salman R., Berger, Dustin R., Ware, Brenton R.
• Primary Examiner(s): Moloye, Titilayo
• Application Number:
  US16/157,930
• Publication Number:
  US 20190048315A1
• Time in Patent Office: 649 Days
• CPC Class Codes: C12N 2501/237 Oncostatin M [OSM]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/067 Hepatocytes

Abstract

The present disclosure provides co-cultures of human pluripotent stem cell derived hepatocytes and at least one non-parenchymal cell population in vitro, methods of preparing the co-cultures and methods of using the co-cultures for high throughput screening and evaluation of drug candidates. The stem cell derived hepatocyte co-culture system provides an in vitro model in which cell viability and relatively mature hepatocyte phenotype of stem cell derived hepatocytes are maintained for extended periods relative to conventional monoculture.

Claims

1. A method of determining the hepatotoxicity of a test compound, the method comprising:

obtaining a co-culture of a population of human hepatocytes derived from induced human pluripotent stem cells and a population of 3T3-J2 fibroblasts in vitro with a layer of material comprising a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells disposed on the coculture;

contacting the co-culture with the test compound;

maintaining the co-culture for a time and under conditions sufficient to allow an effect of the test compound on the human hepatocytes;

measuring at least one indicator of hepatic function in the co-culture to obtain a test measurement, wherein the at least one indicator of hepatic function is albumin production, urea production, or any combination thereof; and

comparing the test measurement to a control measurement from the co-culture before contact with the test compound, wherein the test compound is hepatotoxic if (a) albumin production is significantly decreased in test measurements as compared to the control measurement and/or (b) urea production is significantly decreased in test measurements as compared to the control measurement,

wherein the cell populations are disposed in a micropattern on a culture substrate and the micropattern comprises a predetermined two-dimensional pattern of multiple microdots, the micropattern defined by a microdot diameter and a center-to-center spacing between each of any two neighboring microdots, wherein each microdot has a diameter of about 500 μ

m and the center-to-center spacing between each of any two neighboring microdots is about 1200 μ

m, and the microdots comprise the human hepatocytes derived from induced human pluripotent stem cells and the space between the microdots comprises the 3T3-J2 fibroblast population.

2. The method of claim 1, wherein the culture substrate comprises a glass surface, a polystyrene surface, or a silicon surface.

3. The method of claim 1, wherein the population of human hepatocytes derived from induced human pluripotent stem cells is derived from a population of previously cryopreserved induced human pluripotent stem cell derived hepatocytes.