Expansion and differentiation of human embryonic stem cells to endoderm progeny in a microcarrier stirred-suspension culture

Abstract

Embryonic stem cells (ESCs) with their abilities for extensive proliferation and multi-lineage differentiation can serve as a renewable source of cellular material in regenerative medicine. However, the development of processes for large-scale generation of human ESCs (hESCs) or their progeny will be necessary before hESC-based therapies become a reality. We hypothesized that microcarrier stirred-suspension bioreactors characterized by scalability, straightforward operation, and tight control of the culture environment can be used for hESC culture and directed differentiation. Under appropriate conditions, the concentration of hESCs cultured in a microcarrier bioreactor increased 34- to 45-fold over 8 days. The cells retained the expression of pluripotency markers such as OCT3/4A, NANOG, and SSEA4, as assessed by quantitative PCR, immunocytochemistry, and flow cytometry. We further hypothesized that hESCs on microcarriers can be induced to definitive endoderm (DE) when incubated with physiologically relevant factors. In contrast to embryoid body cultures, all hESCs on microcarriers are exposed to soluble stimuli in the bulk medium facilitating efficient transition to DE. After reaching a peak concentration, hESCs in microcarrier cultures were incubated in medium containing activin A, Wnt3a, and low concentration of serum. More than 80% of differentiated hESCs coexpressed FOXA2 and SOX17 in addition to other DE markers, whereas the expression of non-DE genes was either absent or minimal. We also demonstrate that the hESC-to-DE induction in microcarrier cultures is scalable. Our findings support the use of microcarrier bioreactors for the generation of endoderm progeny from hESCs including pancreatic islets and liver cells in therapeutically useful quantities.

FIG. 1.

Culture of hESCs in a microcarrier suspension. (A) H9 cells were seeded at 5 × 10⁴ (), 10 × 10⁴ (), and 20 × 10⁴ () hESCs/mL and cultured at 45 rpm. (B) Human ESCs were seeded at 1 × 10⁵ cells/mL and propagated at 45 (), 60 (), and 80 () rpm. (C) Colonization of beads by hESCs cultured at 60 rpm. (D) FDA/PI staining of hESCs on beads at 45 rpm (day 8). (E) Cumulative LDH activity for hESCs cultured at 45 rpm. Bars in (D): 50 μm. Color images available online at www.liebertonline.com/ten.

FIG. 1.

Culture of hESCs in a microcarrier suspension. (A) H9 cells were seeded at 5 × 10⁴ (), 10 × 10⁴ (), and 20 × 10⁴ () hESCs/mL and cultured at 45 rpm. (B) Human ESCs were seeded at 1 × 10⁵ cells/mL and propagated at 45 (), 60 (), and 80 () rpm. (C) Colonization of beads by hESCs cultured at 60 rpm. (D) FDA/PI staining of hESCs on beads at 45 rpm (day 8). (E) Cumulative LDH activity for hESCs cultured at 45 rpm. Bars in (D): 50 μm. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Human ESCs expanded on microcarriers retain the expression of pluripotency markers. The expression profile of cultured hESCs was analyzed by (A) RT-PCR, (B) qPCR, (C–E) immunocytochemistry, and (F, G) flow cytometry. In (A) gene expression suggestive of tri-lineage differentiation was minimal. Black curves in (F, G) represent controls. The data shown were obtained from samples from a representative run at 45 rpm. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Human ESCs expanded on microcarriers retain the expression of pluripotency markers. The expression profile of cultured hESCs was analyzed by (A) RT-PCR, (B) qPCR, (C–E) immunocytochemistry, and (F, G) flow cytometry. In (A) gene expression suggestive of tri-lineage differentiation was minimal. Black curves in (F, G) represent controls. The data shown were obtained from samples from a representative run at 45 rpm. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Combined expansion and differentiation of hESCs in a microcarrier bioreactor. (A) H9 hESCs were seeded at 10⁵ hESCs/mL and expanded at 45 rpm for 8 days. On day 8, CM was replaced with differentiation medium. The cell concentration and viability during culture are shown. (B) Cumulative LDH activity corresponding to the culture in (A).

FIG. 4.

Gene expression dynamics of hESCs subjected to DE differentiation in a microcarrier bioreactor. (A) Pluripotency genes were downregulated, whereas (B) DE gene expression increased during differentiation. Expression on day 0(A) or day 8 (B) was set to 1. (C) The expression of DE and non-DE genes is shown for hESCs directed toward DE and for hESCs treated with differentiation medium but without activin A/Wnt3a (control). *p < 0.01 for the expression of corresponding genes compared to that on day 0.

FIG. 5.

DE proteins in differentiated H9 cells. (A, B) Cells on microbeads expressed FOXA2 and SOX17. The SOX17⁺/FOXA2⁺ cell fraction was determined for hESCs on microcarriers treated in differentiation medium (C) with or (D) without activin/Wnt3a. (E) H9 cells differentiated to DE in dishes. SOX17⁺/BRACHYURY⁺ cells after (F) 24 h and (G) 4 days of differentiation in a microcarrier bioreactor culture. Bars in (A): 100 μm. Color images available online at www.liebertonline.com/ten.

FIG. 5.

DE proteins in differentiated H9 cells. (A, B) Cells on microbeads expressed FOXA2 and SOX17. The SOX17⁺/FOXA2⁺ cell fraction was determined for hESCs on microcarriers treated in differentiation medium (C) with or (D) without activin/Wnt3a. (E) H9 cells differentiated to DE in dishes. SOX17⁺/BRACHYURY⁺ cells after (F) 24 h and (G) 4 days of differentiation in a microcarrier bioreactor culture. Bars in (A): 100 μm. Color images available online at www.liebertonline.com/ten.

FIG. 6.

Scalable differentiation of hESCs to DE in a microcarrier bioreactor. (A) Concentration and viability of H9 cells expanded in two bioreactors for 8 days and then subjected to differentiation. On day 9 the cells from the two bioreactors were combined into a single vessel. (B) Cumulative LDH activity. (C) SOX17/FOXA2 coexpression on day 12.

FIG. 6.

Scalable differentiation of hESCs to DE in a microcarrier bioreactor. (A) Concentration and viability of H9 cells expanded in two bioreactors for 8 days and then subjected to differentiation. On day 9 the cells from the two bioreactors were combined into a single vessel. (B) Cumulative LDH activity. (C) SOX17/FOXA2 coexpression on day 12.