In Vitro Differentiation and Maturation of Human Embryonic Stem Cell into Multipotent Cells

Table 3

In vitro models for direct differentiation of embryonic stem cells into osteogenic lineages.

Days in EB formation	Differentiation conditions	Selection	Osteoblastic markers studied	Cells line(s)	Reference
1–6	Directed differentiation in presence of osteogenic factors	Serum and osteogenic factors	D21/35: mineralization nodules, ALP, osteocalcin, and type I collagen	Human-H9, mouse ESCs	[32–34]
7 RA from day 2	RA treated EB outgrowth culture was treated with BMP plus osteogenic factors	RA and BMP-4	D12: mineralization	Mouse-CGR8, E14Tg2a, EFC1	[16]
3	Coculture of EBs with primary bone-derived cells (hPBDs) for 14 days, in vivo bone formation with BMP-2	Conditioned media from hPBDs	D7 and 14: mineralization, osteocalcin, collagen I, osteopontin, BSP	Human-CHA3	[35]
5	The differentiation of single cells was initiated with bioactive glass	Serum and osteogenic factors	D21: mineralization, osteocalcin, ALP, and cbfa-1/Runx2	Mouse-E14	[36]
4-5	Single cell suspension of EB was directly differentiated in presence of osteogenic factors	Serum, osteogenic factors and MACS sorting^#	D21: mineralization, osteocalcin, Cadherin-11, and cbfa-1/Runx2	Human-H1, H9 and Mouse-CEE	[37–39]

Culture medium was α-minimal essential medium or Dulbecco’s modified Eagle’s medium, supplemented with 10–20% foetal calf serum. Factors added for osteogenic differentiation: β-gly, 10 mM (range, 2–10 mM); ascorbic acid, 50 μg/mL; Dex, 10 or 100 nM; RA, 10⁻⁷ M; BMP, 100 ng/mL (range, 10–800 ng/mL). Cbfa1, BSP, ALP, and OC were analyzed by PCR.
^#In the mouse study cells were sorted by Cadherin-11 expression, and cDNA microarray was performed.
ALP: alkaline phosphatase; asc. Acid: ascorbic acid; BMP: bone morphogenetic protein; BSP: bone sialoprotein; Runx2: core binding factor α1; D: day(s); Dex: dexamethasone; ES: embryonic stem; FGF: fibroblast growth factor; β-gly: β-glycerophosphate; RA: retinoic acid; OC: osteocalcin.