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

• US 10,266,806 B2
• Filed: 01/14/2015
• Issued: 04/23/2019
• Est. Priority Date: 01/14/2014
• Status: Active Grant

• Current Assignee: Colorado State University Research Foundation (Colorado State University System)
• Original Assignee: Colorado State University Research Foundation (Colorado State University System)
• Inventors: Khetani, Salman R., Berger, Dustin R., Ware, Brenton R.
• Primary Examiner(s): Moloye, Titilayo
• Application Number:
  US14/596,787
• Publication Number:
  US 20150240203A1
• Time in Patent Office: 1,560 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 composition comprising:

a population of human hepatocytes derived from induced human pluripotent stem cells and a population of 3T3-J2 fibroblasts in co-culture (population of hepatocytes in co-culture) in vitro;

a culture substrate, wherein the cell populations are disposed in a micropattern on the 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; and

a layer of material comprising gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells disposed on the co-culture,

wherein the population of hepatocytes in co-culture exhibits a higher level of cytochrome P450 3A4 (CYP3A4) enzyme activity by at least day 22 of culture as compared to a population of human hepatocytes derived from induced pluripotent stem cells and not co-cultured with a population of 3T3-J2 fibroblasts (population of hepatocytes not in co-culture).

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

3. The composition of claim 1, wherein the population of hepatocytes in co-culture further exhibits a higher level of differentiation toward an adult human hepatocyte phenotype as determined by measuring at least one of liver-specific gene expression, albumin secretion, urea synthesis, Phase I enzyme activity, Phase II enzyme activity, low density lipoprotein (LDL) uptake, and number of active bile canaliculi in the co-cultured hepatocytes and comparing the measurement to that obtained in the population of hepatocytes not in co-culture.

4. The composition 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 pluripotent stem cell derived hepatocytes.

7. The composition of claim 4, wherein the wherein the population of hepatocytes in co-culture exhibits a higher level of cytochrome P450 3A4 (CYP3A4) enzyme activity by at least day 15 of culture as compared to the population of hepatocytes not in co-culture.

8. The composition of claim 1, wherein the wherein the population of hepatocytes in co-culture exhibits a higher level of cytochrome P450 3A4 (CYP3A4) enzyme activity by at least day 15 of co-culture as compared to the population of hepatocytes not in co-culture.

9. The composition of claim 1, wherein the population of hepatocytes not in co-culture is not disposed in a micropattern on the culture substrate.

10. The composition of claim 3, wherein the higher level of differentiation toward an adult human hepatocyte phenotype is determined by measuring albumin secretion, wherein the population of hepatocytes in co-culture exhibits a higher level of albumin secretion as compared to the population of hepatocytes not in co-culture.

11. The composition of claim 3, wherein the higher level of differentiation toward an adult human hepatocyte phenotype is determined by measuring Phase II enzyme activity and wherein the population of hepatocytes in co-culture exhibits a higher level of Phase II enzyme activity by at least day 15 of culture as compared to the population of hepatocytes not in co-culture.

12. The composition of claim 3, wherein the higher level of differentiation toward an adult human hepatocyte phenotype is determined by measuring Phase I enzyme activity, wherein the Phase I enzyme is at least one of cytochrome P1A2 (CYP1A2), cytochrome P2B6 (CYP2B6), and cytochrome 2D6 (CYP2D6), and wherein the Phase I enzyme activity in the population of hepatocytes in co-culture is higher than the Phase I enzyme activity in the population of hepatocytes not in co-culture.

13. The composition of claim 1, wherein the population of hepatocytes in co-culture exhibits a higher level of gene expression of at least one of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) as compared to the population of hepatocytes not in co-culture, wherein the higher level of gene expression is determined by measuring the mRNA transcripts for at least one of PXR and CAR in the population of hepatocytes in co-culture and comparing the measurement to that obtained in the population of hepatocytes not in co-culture.

14. The composition of claim 1, wherein the population of hepatocytes in co-culture exhibits a higher ratio of cytochrome 3A4 (CYP3A4) mRNA transcripts to cytochrome 3A7 (CPY3A7) mRNA transcripts by at least day 16 of culture as compared to the population of hepatocytes not in co-culture.

15. The composition of claim 1, wherein the population of hepatocytes in co-culture exhibits higher cytochrome P450 (CYP450) induction as compared to the population of hepatocytes not in co-culture, when exposed to a drug.

16. The composition of claim 15, wherein the cytochrome P450 (CYP450) induction is determined by an increase in cytochrome P2C9 (CYP2C9) activity in the population of hepatocytes in co-culture as compared to the population of hepatocytes not in co-culture.

17. The composition of claim 16, wherein the drug is selected from rifampicin and phenobarbital.

18. The composition of claim 1, wherein the cytochrome P450 3A4 (CYP3A4) enzyme activity is determined by using a luminescence-based assay.

19. The composition of claim 18, wherein the wherein the population of hepatocytes in co-culture exhibits a higher level of cytochrome P450 3A4 (CYP3A4) enzyme activity by at least day 15 of co-culture as compared to the population of hepatocytes not in co-culture.

20. The composition of claim 18, wherein the population of human hepatocytes derived from induced human pluripotent stem cells is derived from a population of previously cryopreserved induced pluripotent stem cell derived hepatocytes.

21. The composition of claim 20, wherein the wherein the population of hepatocytes in co-culture exhibits a higher level of cytochrome P450 3A4 (CYP3A4) enzyme activity by at least day 15 of culture as compared to the population of hepatocytes not in co-culture.

22. The composition of claim 18, wherein the population of hepatocytes in co-cultured further exhibits a higher level of differentiation toward an adult human hepatocyte phenotype as determined by measuring at least one of liver-specific gene expression, albumin secretion, urea synthesis, Phase I enzyme activity, Phase II enzyme activity, LDL uptake, and number of active bile canaliculi in the co-cultured hepatocytes and comparing the measurement to that obtained in the population of hepatocytes not in co-culture.

23. The composition of claim 22, wherein the higher level of differentiation toward an adult human hepatocyte phenotype is determined by measuring albumin secretion, wherein the population of hepatocytes in co-culture exhibits a higher level of albumin secretion as compared to the population of hepatocytes not in co-culture.

24. The composition of claim 22, wherein the higher level of differentiation toward an adult human hepatocyte phenotype is determined by measuring Phase II enzyme activity and wherein the population of hepatocytes in co-culture exhibits a higher level of Phase II enzyme activity by at least day 15 of culture as compared to the population of hepatocytes not in co-culture.

25. The composition of claim 22, wherein the higher level of differentiation toward an adult human hepatocyte phenotype is determined by measuring Phase I enzyme activity, wherein the Phase I enzyme is at least one of cytochrome P1A2 (CYP1A2), cytochrome P2B6 (CYP2B6), and cytochrome 2D6 (CYP2D6), and wherein the Phase I enzyme activity in the population of hepatocytes in co-culture is higher than the Phase I enzyme activity in the population of hepatocytes not in co-culture.

26. The composition of claim 22, wherein the population of hepatocytes in co-culture exhibits a higher level of gene expression of at least one of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) as compared to the population of hepatocytes not in co-culture, wherein the higher level of gene expression is determined by measuring the mRNA transcripts for at least one of PXR and CAR in the population of hepatocytes in co-culture and comparing the measurement to that obtained in the population of hepatocytes not in co-culture.

27. The composition of claim 18, wherein the population of hepatocytes in co-culture exhibits a higher ratio of cytochrome 3A4 (CYP3A4) mRNA transcripts to cytochrome 3A7 (CPY3A7) mRNA transcripts by at least day 16 of culture as compared to the population of hepatocytes not in co-culture.

28. The composition of claim 18, wherein the population of hepatocytes in co-culture exhibits higher cytochrome P450 (CYP450) induction as compared to the population of hepatocytes not in co-culture, when exposed to a drug.

29. The composition of claim 28, wherein the cytochrome P450 (CYP450) induction is determined by an increase in cytochrome P2C9 (CYP2C9) activity in the population of hepatocytes in co-culture as compared to the population of hepatocytes not in co-culture.

30. The composition of claim 29, wherein the drug is selected from rifampicin and phenobarbital.

5. A method of culturing a population of hepatocytes derived from induced pluripotent human stem cells in vitro comprising:

co-culturing the population of hepatocytes derived from induced pluripotent stem cells with a population of 3T3-J2 fibroblasts and maintaining the co-culture for at least about 8 days, 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 cell population, wherein a layer of material comprising gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells is disposed on the co-culture, and wherein the population of hepatocytes in co-culture exhibits a higher level of cytochrome P450 3A4 (CYP3A4) enzyme activity by at least day 22 of culture as compared to a population of human hepatocytes derived from induced pluripotent stem cells and not co-cultured with a population of 3T3-J2 fibroblasts.

6. The method of claim 5, wherein the culture substrate comprises a glass surface, a polystyrene surface, or a silicon surface.