Inducing human parthenogenetic embryonic stem cells into islet‑like clusters

Abstract

In order to determine whether human parthenogenetic embryonic stem (hpES) cells have the potential to differentiate into functional cells, a modified four‑step protocol was used to induce the hpES cells into islet‑like clusters (ILCs) in vitro. Growth factors activin A, retinoic acid, nicotinamide, Exendin‑4 and betacellulin were added sequentially to the hpES cells at each step. The terminally differentiated cells were shown to gather into ILCs. Immunohistochemistry and semi quantitative polymerase chain reaction analyses demonstrated that the ILCs expressed islet specific hormones and functional markers. Furthermore, an insulin release test indicated that the clusters had the same physiological function as islets. The ILCs derived from hpES cells shared similar characteristics with islets. These results indicate that hpES cell‑derived ILCs may be used as reliable material for the treatment of type I diabetes mellitus.

Figure 1

Schematic diagram of the four-step method used to induce human parthenogenetic embryonic stem cells into islet-like clusters. The differentiation protocol was divided into four steps and the growth factors, medium and length of duration for each step are shown. The black box represents the removal of Activin A and serum followed by culturing the cells in RPMI-1640 medium alone for 10 hours. RPMI 1640, Roswell Park Memorial Institute 1640 medium; DMEM/F12, Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F12; NIC, nicotinamide; RA, retinoic acid; d, day; h, hour.

Figure 2

Generation of DE cells. (A) Expression levels of Sox17 on day 5 in the three embryonic stem cell lines.(B) DE marker sox17 was detected on day 5 by immunofluorescence analysis. The nuclei were stained with DAPI. Data are expressed as the mean ±standard deviation. Scale bar: 100 μm. chHES8, normal human ES; chHES32 and chHES69, human parthenogenetic ES; ES, embryonic stem cells; DE, definitive endoderm.

Figure 3

Differentiation of PP cells. (A–C) Effects of RA in pancreatic specialization. (A) Gene expression in response to RA treatment at different concentrations for 24 h following Activin A treatment for 5 days and 10 h treatment with L-glutamine. (B) Gene expression in response to 24 h 10 6 M RA treatment at different time intervals. (C) Gene expression in response to 24 and 48 h treatments with 10⁻⁵ M and 10⁻⁶ M RA for 10 h. (D) Expression levels of Pdx1 on day 13 in the three embryonic stem cell lines. (E) PP marker Pdx1 was detected on day 13 by immunofluorescence analysis. The nuclei were stained with DAPI. Data are expressed as the mean ± standard deviation. Scale bar: 50um. ES, embryonic stem cells; PP, pancreatic precursor; RA, retinoic acid.

Figure 4

Expression of marker genes, mesoderm and ectoderm-related genes, and imprinted genes on days 0, 5, 6 and 13, as determined by quantitative polymerase chain reaction. (A) The expression levels of marker genes for embryonic stem cells (ESCs) (Oct4, Nanog), definitive endoderm (Sox17, Foxa2, Cxcr4) and pancreatic precursors (Pdx1, Hb9, Hnf6) in the three embryonic stem cell lines. (B) The expression levels of marker genes of the mesoderm (Brachyury, Flk, Scl1, Runx1) and ectoderm (Pax6, Sox1, Krt17) in the three embryonic stem cell lines. (C) The expression levels of paternally imprinted (IGF2, SNP1, SNPRN, IPW), maternally imprinted (H19, CDKNIC, NES55), and receptor (IGF1R, IGF2R) genes on days 5, 13 and 20, in the three embryonic stem cell lines. DE, defined endoderm; chHES8, normal human ESCs; chHES32, human parthenogenetic ESCs; chHES69, human parthenogenetic ESCs.

Figure 5

Identification of insulin producing cells. (A) Expression levels of imprinted genes and receptor genes on day 20 in the three embryonic stem cell lines, as determined by qPCR. (B) Expression levels of insulin on day 20. in the three embryonic stem cell lines. (C) Islets marker insulin was detected on day 20 by immunofluorescence analysis. Nuclei were stained with DAPI. Scale bar: 50 μm. (D) Insulan release in response to a glucose stimulus of 5.5 and 25mM in the three embryonic stem cell lines, as determined by an insulin release assay. (E) Expression levels of imprinted genes and receptor genes on day 20 in the theee embryonic stem cell lines, as determined by semi qPCR. Data are expressed as the mean ± standard deviation. Statistical analyses were performed using Student’s t-test. ^*P<0.05. chHES8 normal human ESCs; chHES32 and chHES69, human parthenogenetic embryonic stem cells; qPCR, quantitative polymerase chain reaction.

Figure 6

Proliferative ability assay. (A) Expression efficiency of Ki67 on days 5, 13 and 20, in the three embryonic stem cell lines. (B) Immunohistochemistry of the Ki67 protein on days 5, 13 and 20 visualized using DAPI staining techniques. chHES8, normal human ESCs; chHES32 and chHES69, human parthenogenetic embryonic stem cells. Scale bar: 50 μm. Data are expressed as the mean ± standard deviation. Statistical analyses were performed using Student’s t-test. *P<0.05.. chHES8 normal human ESCs; chHES32 and chHES69, human parthenogenetic embryonic stem cells.