Enhanced Differentiation of Stromal Cells and Embryonic Stem Cells with Vitamin D3

In-vitro mouse co-culture of E14 embryonic stem cells
(ESCs) and OP9 stromal cells can recapitulate the earliest stages of
haematopoietic development, not accessible in human embryos,
supporting both haemogenic precursors and their primitive
haematopoietic progeny. 1α, 25-Dihydroxy-vitamin D3 (VD3) has
been demonstrated to be a powerful differentiation inducer for a wide
variety of neoplastic cells, and could enhance early differentiation of
ESCs into blood cells in E14/OP9 co-culture. This study aims to
ascertain whether VD3 is key in promoting differentiation and
suppressing proliferation, by separately investigating the effects of
VD3 on the proliferation phase of the E14 cell line and on stromal
OP9 cells.The results showed that VD3 inhibited the proliferation of
the cells in a dose-dependent manner, quantitatively by decreased cell
number, and qualitatively by alkaline-phosphatase staining that
revealed significant differences between VD3-treated and untreated
cells, characterised by decreased enzyme expression (colourless
cells). Propidium-iodide cell-cycle analyses showed no significant
percentage change in VD3-treated E14 and OP9 cells within their G
and S-phases, compared to the untreated controls, despite the
increased percentage of G-phase compared to the S-phase in a dosedependent
manner. These results with E14 and OP9 cells indicate that
adequate VD3 concentration enhances cellular differentiation and
inhibits proliferation. The results also suggest that if E14 and OP9
cells were co-cultured andVD3-treated, there would be furtherenhanced
differentiation of ESCs into blood cells.

• Mayada Alqaisi
• Nasser Al-Shanti
• Quiyu Wang
• William S. Gilmore

• Differentiation
• embryonic stem cells
• OP9 stromal cells
• 1α
• 25-dihydroxy-vitamin D3

<p>[1] M. J. Evans and M. H. Kaufman, "Establishment in culture of
pluripotential cells from mouse embryos," Nature, vol. 292, pp. 154-156,
1981.
[2] S.-I. Nishikawa, L. M. Jakt, and T. Era, "Embryonic stem-cell culture as
a tool for developmental cell biology," Nature Reviews Molecular Cell
Biology, vol. 8, pp. 502-507, 2007.
[3] M. A. Vodyanik, J. A. Bork, J. A. Thomson, and I. I. Slukvin, "Human
embryonic stem cell–derived CD34+ cells: efficient production in the
coculture with OP9 stromal cells and analysis of lymphohematopoietic
potential," Blood, vol. 105, pp. 617-626, 2005.
[4] H. F. DeLuca, "Overview of general physiologic features and functions
of vitamin D," The American journal of clinical nutrition, vol. 80, pp.
1689S-1696S, 2004.
[5] N. I. zurNieden, F. D. Price, L. A. Davis, R. E. Everitt, and D. E.
Rancourt, "Gene profiling on mixed embryonic stem cell populations
reveals a biphasic role for β-catenin in osteogenic differentiation,"
Molecular Endocrinology, vol. 21, pp. 674-685, 2007.
[6] P. H. Anderson, B. May, and H. A. Morris, "Vitamin D metabolism:
new concepts and clinical implications," The Clinical Biochemist
Reviews, vol. 24, p. 13, 2003.
[7] A. Grande, M. Montanari, E. Tagliafico, R. Manfredini, T. Z. Marani,
M. Siena, et al., "Physiological levels of 1α, 25 dihydroxyvitamin D3
induce the monocytic commitment of CD34+ hematopoietic
progenitors," Journal of leukocyte biology, vol. 71, pp. 641-651, 2002.
[8] H. F. Luderer and M. B. Demay, "The vitamin D receptor, the skin and
stem cells," The Journal of Steroid Biochemistry and Molecular Biology,
vol. 121, pp. 314-316, 2010.
[9] T. C. Chen, G. G. Schwartz, K. L. Burnstein, B. L. Lokeshwar, and M.
F. Holick, "The in vitro evaluation of 25-hydroxyvitamin D3 and 19-
nor-1α, 25-dihydroxyvitamin D2 as therapeutic agents for prostate
cancer," Clinical Cancer Research, vol. 6, pp. 901-908, 2000.
[10] J. O’Kelly, J. Hisatake, Y. Hisatake, J. Bishop, A. Norman, and H. P.
Koeffler, "Normal myelopoiesis but abnormal T lymphocyte responses
in vitamin D receptor knockout mice," Journal of Clinical Investigation,
vol. 109, pp. 1091-1099, 2002.
[11] S. Yetgin and S. Yalçin, "The effect of vitamin D3 on CD34 progenitor
cells in vitamin D deficiency rickets," TURKISH JOURNAL OF
PEDIATRICS, vol. 46, pp. 164-166, 2004.
[12] H. Okuno, K. N. Kishimoto, M. Hatori, and E. Itoi, "1α,
25-dihydroxyvitamin D3 enhances fast