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

Table 1

In vitro models for direct differentiation of embryonic stem cells to chondrogenic lineages.

Days in EB formation	Post EB differentiation	Differentiation conditions	Chondrogenic markers studied	Cells line(s)	Reference
5	Encapsulation of EBs in PEG hydrogel for 17 days	The EBs in hydrogel were differentiated in presence of chondrogenic medium (CM) treated with BMP-2 and TGF-β1	Type I, II, X collagen, and osteocalcin. Proteoglycans content measurement	Mouse-D3 and human-BG03	[12–15]
N/R	EB cells were seeded on ceramic particles	Cells on the ceramic scaffold was differentiated in vitro for 21 days in CM supplemented with TGF-β3 and 21 days of in vivo maturation	Morphology was studied and proteoglycans content was analyzed	Mouse-IB10, human MSC, and Goat MSCs	[4]
7 RA from day 2	EBs were plated	RA-treated EB outgrowth culture was treated with TGF-β3	D15: proteoglycans, Col2a1, Sox9, Col10a1 and MMP13	Mouse-CGR8, E14Tg2a, EFC1	[16]
28	After 4 weeks the EBs were dissociated and self-assembled for additional 4 weeks	EBs were differentiated in chondrogenic medium supplemented with combinations of TGF-β1, TGF-β3, BMP-2, and IGF-1 (100 ng/mL)	Sox9, Col1a1, Col2a1	Human-BG01V	[17]

Culture medium was high glucose Dulbecco’s modified Eagle’s medium supplemented with 1–5% foetal calf serum. Factors added for chondrocyte differentiation: ascorbic acid, 50 μg/mL; Dex, 10 or 100 nM; proline, 40 μg/mL; sodium pyruvate, 100 μg/mL; ITS, 50 mg/mL; TGF-β, 10 ng/mL; RA, 10⁻⁷ M; BMP, 10 ng/mL (range, 10–800 ng/mL). Aggrecan, Sox9, type II collagen, type X collagen, and scleraxis were analyzed by PCR.
BMP: bone morphogenetic protein; Col: collagen; D: day(s); Dex: dexamethasone; EB: embryoid body; FGF: fibroblast growth factor; IGF: insulin-like growth factor; MMP: Matrix metalloproteases; PEG: poly-ethylene glycerol; RA: retinoic acid; TGF-β: transforming growth factor-β; OC: osteocalcin.