Int J Stem Cells.  2019 Jul;12(2):240-250. 10.15283/ijsc18137.

Comparative Evaluation of Hormones and Hormone-Like Molecule in Lineage Specification of Human Induced Pluripotent Stem Cells

• Choi SA
• An JH
• Lee SH
• Lee GH
• Yang HJ
• Jeong PS
• Cha JJ
• Lee S
• Park YH
• Song BS
• Sim BW
• Kim YH
• Kim JS
• Jin YB
• Huh JW
• Lee SR
• Lee JH
• Kim SU

• KMID: 2465895
• DOI: http://doi.org/10.15283/ijsc18137

Abstract

BACKGROUND AND OBJECTIVES
Proficient differentiation of human pluripotent stem cells (hPSCs) into specific lineages is required for applications in regenerative medicine. A growing amount of evidences had implicated hormones and hormone-like molecules as critical regulators of proliferation and lineage specification during in vivo development. Therefore, a deeper understanding of the hormones and hormone-like molecules involved in cell fate decisions is critical for efficient and controlled differentiation of hPSCs into specific lineages. Thus, we functionally and quantitatively compared the effects of diverse hormones (estradiol 17-β (E2), progesterone (P4), and dexamethasone (DM)) and a hormone-like molecule (retinoic acid (RA)) on the regulation of hematopoietic and neural lineage specification.
METHODS AND RESULTS
We used 10 nM E2, 3 μM P4, 10 nM DM, and 10 nM RA based on their functional in vivo developmental potential. The sex hormone E2 enhanced functional activity of hematopoietic progenitors compared to P4 and DM, whereas RA impaired hematopoietic differentiation. In addition, E2 increased CD34⁺CD45⁺ cells with progenitor functions, even in the CD43⁻ population, a well-known hemogenic marker. RA exhibited lineage-biased potential, preferentially committing hPSCs toward the neural lineage while restricting the hematopoietic fate decision.
CONCLUSIONS
Our findings reveal unique cell fate potentials of E2 and RA treatment and provide valuable differentiation information that is essential for hPSC applications.

Keyword

Human induced pluripotent stem cells; lineage specification; Estradiol-17β; Retinoic acid; Hematopoietic differentiation; Cell fate decision

MeSH Terms

Dexamethasone
Humans*
Induced Pluripotent Stem Cells*
Pluripotent Stem Cells
Progesterone
Regenerative Medicine
Tretinoin
Dexamethasone
Progesterone
Tretinoin

Figure

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  Fig. 1 Derivation of hiPSCs from BM CD34+ cells. (A) Representative phase-contrast image of hiPSCs. hiPSCs were characterized by embryonic stem cell morphology. Scale bar, 200 μm. (B) Immunocytochemical analysis of pluripotent markers. Representative images of hiPSCs stained with the pluripotent markers OCT3/4, NANOG, SOX2, SSEA3, SSEA4 and TRA-1-60. Scale bar, 200 μm. Nuclei were stained with hoechst33342 (blue). (C) Flow cytometric analysis of pluripotent marker expression. Black histogram bars indicate negative isotype controls. (D) Karyotype analysis of hiPSCs, displaying a normal 44+XX karyotype. (E) Quantitative real-time PCR analysis of pluripotent genes in hiPSCs normalized to GAPDH. #Data are presents as the means±SD of three independent experiments. *p<0.05; **p<0.01.

• 

  Fig. 2 Distinct hematopoietic differentiation potential of hormones and hormone-like molecule. (A) Schematic protocol for hematopoietic differentiation of hPSCs. (B~F) Effects of hormones (E2, P4, and DM) and a hormone-like molecule (RA) on the production of hematopoietic cells (CD45+) and progenitors (CD34+CD45+). Ctrl: control, E2: estradiol-17β, P4: progesterone, DM: dexamethasone. (B) Flow cytometric plots showing hematopoietic cells (CD45+) and progenitors (CD34+CD45+) at day 12 following hematopoietic differentiation with or without E2, P4, DM, and RA treatment. (C) Relative levels of CD45+ cells normalized to the control. The inset shows the percentage of CD45+ cells. (D) Relative levels of hematopoietic progenitors (CD34+CD45+) normalized to the control. The inset indicates the percentage of CD34+CD45+ cells. Number of (E) total cells and (F) CD45+ cells during hematopoietic differentiation with or without treatment with hormones and hormone-like molecule. (G) Total number of hematopoietic CFUs generated from day 12 hEBs. (H) Distribution of colony subtypes derived from day 12 hEBs. (I) Representative CFU morphology. Scale bar, 100 μm. CFU-granulocytes (CFU-G); CFU-macrophages (CFU-M); CFU-granulocytes/macrophages (CFU-GM); CFU-erythroid (CFU-E). #Data are presents as the means±SD of three or six independent experiments. *p<0.05; **p<0.01.

• 

  Fig. 3 E2 enhances the functional ability of hemogenic precursors. (A) i. Flow cytometric analysis identifying the CD43+ population during hematopoietic differentiation. ii. Relative levels of CD43+ cells normalized to the control. The inset shows the percentage of CD43+ cells. (B) Flow cytometry gating of the CD43+ and CD43− fraction to analyze the frequency of hematopoietic progenitors (CD34+CD45+). (C) Relative proportions of hematopoietic progenitors of CD43+ fractions normalized to the control at day 12. The inset represents the percentage of CD34+CD45+ cells. (D) Relative levels of CD34+CD45+ cells gated from the CD43− population normalized to the control. The inset displays the percentage of CD34+CD45+ cells. (E) Representative CFU morphologies derived from the CD43−CD34+CD45+ population treated with E2. Scale bar, 100 μm. #Data are presents as the means±SD of three independent experiments. *p<0.05; **p<0.01.

• 

  Fig. 4 RA promotes differentiation into the neural lineage. (A) Schematic protocol for neural differentiation of hPSCs. (B~F) Effects of hormones (E2, P4, and DM) and a hormone-like molecule (RA) on the production of neural progenitors (NESTIN+ and PAX6+). (B) Flow cytometric plots showing neural progenitors at days 7~10 following neural differentiation with or without E2, P4, DM, and RA treatment. (C) Relative levels of NESTIN+ cells normalized to the control. The inset shows the percentage of NESTIN+ cells. (D) Relative levels of PAX6+ cells normalized to the control. The inset indicates the frequency of PAX6+ cells. (E) Quantitative real-time PCR analysis of the neural progenitor markers NeuroD and PAX6 and the neural filament marker NF68. Gene expression is normalized to that of GAPDH. (F) Immunocytochemical analysis of βIII-tubulin from control and RA-treated cells during neural differentiation. (G) Schematic diagram of the effect of E2 and RA on lineage specification of hPSCs into hematopoietic and neural lineage. Scale bar, 100 μm. #Data are presents as the means±SD of three independent experiments. *p<0.05; **p<0.01.

Reference

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