Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells

Table 1 Protocols to produce induced pluripotent stem cell-derived mesenchymal stem cells by the mesenchymal stem cell Switch methods

Ref.	iPSC origin	iPSC to iMSC protocol	Time	Application	Citations
Lian et al[41], 2010	Lung fibroblast	(1) iPSC cultured on a gelatinized dish + KO DMEM + 10% SRM + bFGF + PDGFAB + EGF; and (2) FACS SORTING: CD24- and CD105+ and single cell clones plating	7	Limb ischemia in mice	419
Giuliani et al[42], 2011	Amniocytes	iPSC cultured 4 wk in DMEM/F12 + 10% hiFBS + b-FGF + NEAA + L-Glutamine + β-ME + p/s	28	Immunomodulatory properties of iMSC on NK cytolytic activity	110
Liu et al[43], 2012	Dermal fibroblast	(1) iPSC cultured on collagen-coated dishes with α-MEM + 10% FBS + dexamethasone + magnesium L-ascorbic acid phosphate + p/s; and (2) Cells cultured on collagen-coated dishes with α-MEM + 10% FBS + L-Glutamine + NEAA + p/s	10	iMSC generation w/ Fibrillar Collagen Coating	118
Zou et al[44], 2013	Dermal fibroblasts	iPSC medium switched for MSC medium: DMEM-low glucose + 10% FBS + L-Glutamine	14	Generation of osteogenesis 3D scaffolds	98
Hynes et al[45], 2014	Gingival fibroblast periodontal ligaments	(1) iPSC cultured with MSC medium: α-MEM + FCS + sodium pyruvate + l-ascorbate-2-phosphate + L-Glutamine + NEAA + HEPES + p/s; and (2) Cells cultured on gelatin-coated-flasks then switch to non-coated flasks	14	Generation of iMSC	102
Jeong et al[46], 20141	NA	(1) iPSCs cultured in iMSC-inducing medium: DMEM/F12 + 20% KOSR + SB431542 (TGFβ inhibitor); (2) EB grown on matrigel + DMEM/F12 + 0.5% BSA + 10% ITS + SB431542 (TGFβ inhibitor); and (3) Outgrowth grown with DMEM/F12 + 10% FBS + p/s	17	Duchene muscular dystrophy	17
Hu et al[47], 2015	iPS-S-01, C1P33, PCKDSF001C1	iPSC medium switched for MSC medium: DMEM-low glucose + 10% FBS + L-Glutamine, then cultured in gelatin-coated dishes	14	Limb ischemia	177
Kang et al[48], 2015	Dermal fibroblasts	iPSC cultured with MSC medium: DMEM low glucose + FBS 10% + L-Glutamine + p/s then cultured on gelatin-coated dishes	14	iMSC plasticity (less adipogenesis)	65
Zhang et al[23], 2015	PBMCs	(1) iPSC medium switched for MSC medium: DMEM-low glucose + 10% FBS + L-Glutamine + NEAA + p/s; and (2) Cells cultured on gelatin-coated dishes	17	Cutaneous wound healing	262
Lian et al[49], 2016	NA	(1) iPSC cultured on gelatin-coated plates with MSC differentiation medium: KO DMEM + KOSR + bFGF + PDGFAB + EGF; and (2) FACS: CD24- CD105+ cells cultured on gelatin-coated plates with DMEM + 10% FBS + bFGF + PDGFAB + EGF	20	Directed differentiation of iPSC to MSC	15
Gao et al[50], 2017	Urine cell; Amniocytes	(1) iPSC cultured with MSC-inducing culture media: α-MEM + SRM + sodium pyruvate + l-ascorbate-2-phosphate + L-Glutamine + NEAA + p/s on gelatin-coated plates; and (2) Cells cultured with MSC maintenance medium = high-glucose DMEM + 10% FBS + bFGF + EGF	17	iMSC effect on dendritic cells	27
Nachlas et al[51], 20181	NA	(1) iPSC cultured in suspension (to promote cell aggregate) with differentiation media: KO-DMEM + β-ME+ L-Glutamine + 20% FBS + NEAA + p/s, then, cells were cultured on gelatin coated plates; and (2) Cells cultured with iMSC media: KO-DMEM + L-Glutamine + 10% FBS + NEAA + p/s	12	Generation of valve interstitial-like cells from iMSC	14
Wang et al[52], 2018	Amniocytes	(1) iPSC cultured with induction medium: α-MEM + 10% FBS + p/s + L-Glutamine + NEAA + sodium pyruvate + l-ascorbate-2- phosphate; (2) Cells plated on gelatin-coated plates; and (3) Cells plated on uncoated plates with iPSC-MSC medium = High-Glucose DMEM + FBS + bFGF + EGF + p/s	14	Effect of Dexamethasone on iMSC	3
Wang et al[53], 2018	PBMCs	iPSC cultured with MSC medium: Low-Glucose DMEM + 10% FBS + p/s + L-glutamine	NA	Immunomodulatory properties of MSC, transcriptome analysis	8
McGrath et al[54], 20191	Dermal fibroblast	(1) iPSC-MP thawed and expanded in KO DMEM + bFGF + L-Glutamine + MEM + NEAA + 20% FBS + Antibiotic-Antimycotic + β-ME; and (2) Cell are plated into gelatin coated-plates with KO DMEM + heparin + hPL + bFGF + L-Glutamine + MEM NEAA + Antibiotic-Antimycotic + β-ME	NA	iMSC differentiation: GMP-compatible and xeno-free cultivation	6

iPSC origin refers to the cell type used for reprograming; Time is indicated as the minimum number of days required to obtain iMSCs; Citations show numbers on March 2020.

¹In references indicate the study includes methods in more than one protocol category. bFGF: Basic fibroblast growth factor; β-ME: β-Mercaptoethanol; BSA: Bovine serum albumin; CDM: Chemical defined medium; DMEM/F12: Dulbecco's modified Eagle’s medium F12; EB: Embryoid body; EGF: Epidermal growth factor; FBS: Fetal bovine serum; FCS: Fetal calf serum; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; hiFBS: Heat inactivated fetal bovine serum; hPL: human platelet lysate; IMDM: Iscove's modified Dulbecco's media; iMSCs: Induced pluripotent stem cell-derived mesenchymal stem cells; iPSC: Induced pluripotent stem cell; ITS: Insulin-transferrin-selenium; KO-DMEM: Knockout Dulbecco's modified Eagle's medium; KOSR: Knock-out serum replacement; α-MEM: Minimum essential medium Eagle; NA: Not available; NEAA: Non-essential amino acid; p/s: Penicillin-streptomycin; SRM: Serum replacement medium; TGF: Transforming growth factor.

Table 2 Protocols to produce induced pluripotent stem cell-derived mesenchymal stem cells by embryoid bodies approaches

Ref.	iPSC origin	iPSC to iMSC protocol	Time	Application	Citations
Ahfeldt et al[56], 2012	Foreskin fibroblast	(1) iPSC cultured into low-adhesion dishes for EB formation with DMEM + 15% FBS + L-Glutamine; (2) EB plated into gelatin-coated dishes with DMEM + 15% FBS + L-Glutamine; and (3) Cells plated with Mensenchymal Progenitor Cell (MCP) medium: DMEM + 15% FBS + L-Glutamine + bFGF	12	Producing white and brown adipocytes from hPSCs	194
Chen et al[57], 20121	Lung fibroblast	SB431542 inhibitor differentiation method (feeder free); iPSC cultured in inhibitor differentiation medium: KOSR medium + SB431542 (TGFβ inhibitor)Without bFGF to enhance differentiation. Embryoid body differentiation method: (1) EB formation in KOSR medium; and (2) EB cultured with MSC medium: DMEM + 10% FCS + L-Glutamine + gentamicin + p/s	17	Generation of iMSC with TGF-beta inhibitor	136
Villa-Diaz et al[58], 2012	Dermal fibroblasts	(1) EB formation in suspension cultured into ultra-low-attachment plates; and (2) EB plated on gelatin-coated dishes with MSC medium: α-MEM + 10% FBS + L-Glutamine + NEAA + FGF2	21	iMSC from iPSC cultured on synthetic substrate (PMEDSAH)	262
Wei et al[59], 20121	Dermal fibroblasts	(1) EB formation through cardiac differentiation protocol involving cardiomyogenic medium CARM: High-Glucose DMEM + L-Glutamine + NEAA + Selenium Transferrin + β-ME + SB 203580 (p38-MAPK inhibitor); and (2) EB plating on gelatin-coated plates with DMEM + 2% FBS	21	Generation of iMSC	64
Shao et al[60], 2013	MSC	(1) iPSC cultivated in suspension in the differentiation medium: KO DMEM + 20% FBS+ 1% NEAA + β-ME+ L-Glutamine for EB formation; and (2) Embryoid bodies plated on gelatin-coated dishes	19	iMSC DNA methylation profiles	48
Jeong et al[46], 20141	NA	(1) iPSCs cultured in iMSC-inducing medium: DMEM/F12 + 20% KOSR + SB431542 (TGFβ inhibitor); (2) EB grown on matrigel + DMEM/F12 + 0.5% BSA + 10% ITS + SB431542 (TGFβ inhibitor); and (3) Outgrowth grown with DMEM/F12 + FBS (10%) + p/s	17	Duchene muscular dystrophy	17
Miao et al[61], 2014	Dermal fibroblasts	EB cultured with DMEM + 10% FBS	NA	Myocardial infarctus	38
Tang et al[21], 2014	bone marrow	(1) iPSC cultured in ultra-low attachment plate to form EB with differentiation medium: DMEM/F12 + 20% KSR + MEF medium + NEAA + L-Glutamine + β-ME; and (2) EB plated into gelatin-coated plates + MSC growth medium: DMEM + 10% FBS + L-Glutamine + p/s	20	iMSC and calcium phosphate scaffold for bone regeneration	88
Sheyn et al[20], 2016	Dermal fibroblasts	(1) iPSC plated into PCR plates to form EB with IMDM medium: MDM media + KOSR + NEAA + β-ME + PSA antifungal-antibacterial solution; (2) EB transferred to poly-HEMA-coated flasks; (3) Attached EB (aiMSCs) and Transferred EB (tiMSCs) cultured into gelatin-coated flask with medium + TGF-β1; and (4) Medium switched for DMEM + 10% FBS + L-Glutamine + p/s	10	Generation of iMSC and repair bone defect	60
Eto et al[55], 20181	Skin fibroblast	(1) iPSCs treated with CTK (collagenase type IV + 0.25% trypsin + KSR) and transferred to petri dishes to form EB with: DMEM/F12 + 20% KOSR + glutamine + NEAA + BMP4 + p/s; (2) Specific Differentiation or Mesodermal Differentiation: EB cultured on collagen-coated plates + αMEM + 10% FBS + bFGF + BMP4 + Activin A + LiCl + p/s; or Neuroepithelial differentiation: αMEM + 10% FBS + β-ME + RA; and (3) FACS: PDGFR-α+ and VEGFR2+ cells resuspended on collagen-coated plates with αMEM + 10% FBS + 20% KOSR	10	iMSC from mesoderm or neuroepithelium differentiation	7
Nachlas et al[51], 20181	NA	(1) iPSC cultured in suspension (to promote cell aggregate) with differentiation media: KO-DMEM + β-ME + L-Glutamine + 20% FBS + NEAA + p/s, then cells were cultured on gelatin coated plates; and (2) Cells cultured with iMSC media: KO-DMEM + L-Glutamine + 10% FBS + NEAA + p/s	12	Generation of valve interstitial-like cells from iMSC	14
Karam et al[62], 2020	PBMC	(1) EB cultured into ultra-low attachment plates with differentiation medium: Low-Glucose DMEM + 15% FBS + p/s; (2) Later, RA is added to enhance EB formation; (3) EB plating into gelatin/matrigel coated plates + differentiation medium; and (4) Later bFGF is added	14	Generation of iMSC and adipocytes	NA
Huang et al[63], 2020	PBMC	(1) iPSC cultured in suspension to form EB; and (2) Cells plated into gelatin-coated plates with α-MEM + FGF2	NA	Repair of acute kidney injury	NA

iPSC origin refers to the cell type used for reprograming; Time is indicated as the minimum number of days required to obtain iMSCs; Citations show numbers on March 2020.

¹In references indicate the study includes methods in more than one protocol category. bFGF: Basic fibroblast growth factor; β-ME: β-Mercaptoethanol; BMP4: Bone morphogenetic protein 4; BSA: Bovine serum albumin; CDM: Chemical defined medium; DMEM/F12: Dulbecco's modified Eagle’s medium F12; EB: Embryoid body; EGF: Epidermal growth factor; FBS: Fetal bovine serum; FCS: Fetal calf serum; FGF2: Fibroblast growth factor 2; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; hiFBS: Heat inactivated fetal bovine serum; hPL: human platelet lysate; IMDM: Iscove's modified Dulbecco's media; iMSCs: Induced pluripotent stem cell-derived mesenchymal stem cells; iPSC: Induced pluripotent stem cell; ITS: Insulin-transferrin-selenium; KO-DMEM: Knockout Dulbecco's modified Eagle’s medium; KOSR: Knock-out serum replacement; KSR: Knockout serum replacement; α-MEM: Minimum essential medium Eagle; NA: Not available; PBMC: Peripheral blood mononuclear cell; p/s: Penicillin-streptomycin; SRM: Serum replacement medium; TGF: Transforming growth factor; VEGFR: Vascular endothelial growth factor.

Table 3 Protocols to produce induced pluripotent stem cell-derived mesenchymal stem cells by Specific Differentiation approaches

Ref.	iPSC origin	iPSC to iMSC protocol	Time	Application	Citations
Wei et al[59], 20121	Dermal fibroblasts	(1) EB formation through cardiac differentiation protocol involving cardiomyogenic medium CARM: High-Glucose DMEM + L-Glutamine + NEAA + Selenium Transferrin + β-ME + SB 203580 (p38-MAPK inhibitor); and (2) EB plating on gelatin-coated plates with DMEM + 2% FBS	21	Generation of iMSC	64
Fukuta et al[66], 20141	Dermal fibroblast	(1) Induction of hNCC from iPSC; (2) Cells cultured on fibronectin-coated dishes with STK2 medium + CDM (IMDM/Ham's F-12 + lipid concentrate + apo-transferrin + monothioglycerol + BSA + insulin + p/s) + SB431542 (TGFβ inhibitor) + CHIR (Wnt Agonist); and (3) Cells cultured with αMEM + 10% FBS	15	iMSC differentiation through neural crest lineage	80
Ouchi et al[65], 2016	Dermal fibroblast	(1) Generation of NCL (neural crest like-cells); and (2) NCL cultured into DMEM/F12 + Neurobasal medium + L-Glutamine + Gem21 Neuroplex + N2 Supplement + hbFGF + hEGF + insulin + p/s	10	iNCC can develop into iMSC	8
Eto et al[55], 20181	skin fibroblast	(1) iPSCs treated with CTK (collagenase type IV + 0.25% trypsin + KSR) and transferred to petri dishes to form EB with: DMEM/F12 + 20% KOSR + glutamine + NEAA + BMP4 + p/s; (2) Specific Differentiation or Mesodermal Differentiation: EB cultured on collagen-coated plates + αMEM + 10% FBS + bFGF + BMP4 + Activin A + LiCl + p/s or Neuroepithelial differentiation: αMEM + 10% FBS + β-ME + RA; (3) FACS: PDGFR-α+ and VEGFR2+ cells resuspended on collagen-coated plates with αMEM + 10% FBS + 20% KOSR	NA	iMSC from mesoderm or neuroepithelium differentiation	7
Mitsuzawa et al[64], 2019	NA	(1) Induction of hNCC: CDM (IMDM/Ham's F-12 + lipid concentrate + apo-transferrin+ monothioglycerol + BSA + insulin + p/s) + SB431542 (TGFβ inhibitor) + CHIR (Wnt Agonist)Maintenance in DMEM + 10% FBS + FGF2; and (2) Induction of iMSC with DMEM + 10% FBS + FGF2 on fibronectin coated plates	25	Hind limb in rat allotransplantation	1

iPSC origin refers to the cell type used for reprograming; Time is indicated as the minimum number of days required to obtain iMSCs; Citations show numbers on March 2020.

¹In references indicate the study includes methods in more than one protocol category. bFGF: Basic fibroblast growth factor; β-ME: β-Mercaptoethanol; BMP4: Bone morphogenetic protein 4; BSA: Bovine serum albumin; CARM: Cardiomyogenic medium; CDM: Chemical defined medium; CHIR: Wnt agonist, potent GSK3 inhibitor; DMEM/F12: Dulbecco's modified Eagle's medium EB: Embryoid body; FBS: Fetal bovine serum; FGF2: Fibroblast growth factor 2; Ham's F12: Medium formulated for single-cell plating of near-diploid Chinese hamster ovary cells; hbFGF: Human basic fibroblast growth factor; hEGF: Human epidermal growth factor; IMDM: Iscove's modified Dulbecco's media; iMSCs: Induced pluripotent stem cell-derived mesenchymal stem cells; iPSC: Induced pluripotent stem cell; ITS: Insulin-transferrin-selenium; KOSR: Knock-out serum replacement; KSR: Knockout serum replacement; NA: Not available; NEAA: Non-essential amino acid; α-MEM: Minimum essential medium Eagle; PDGFR: Platelet-derived growth factor receptor A; p/s: Penicillin-streptomycin; RA: Retinoic acid; TGF: Transforming growth factor; VEGFR: Vascular endothelial growth factor.

Table 4 Protocols to produce induced pluripotent stem cell-derived mesenchymal stem cells by Pathway Inhibitor approaches

Ref.	iPSC origin	iPSC to iMSC protocol	Time	Application	Citations
Chen YS et al[57], 20121	Lung fibroblast	SB431542 Inhibitor Differentiation Method (feeder free); iPSC cultured in inhibitor differentiation medium: KOSR medium + SB431542 (TGFβ inhibitor)Without bFGF to enhance differentiation; Embryoid body differentiation method: (1) EB formation in KOSR medium; and (2) EB cultured with MSC medium: DMEM + 10% FCS + L-Glutamine + gentamicin + p/s	17	Generation of iMSC with TGF-beta inhibitor	136
Wei et al[59], 20121	Dermal fibroblasts	(1) EB formation through cardiac differentiation protocol involving cardiomyogenic medium CARM: High-Glucose DMEM + L-Glutamine + NEAA + Selenium Transferrin + β-ME + SB 203580 (p38-MAPK inhibitor); and (2) EB plating on gelatin-coated plates with DMEM + 2% FBS	21	Generation of iMSC	64
Fukuta et al[66], 20141	Dermal fibroblast	(1) Induction of hNCC from iPSC; (2) Cells cultured on fibronectin-coated dishes with STK2 medium + CDM (IMDM/Ham's F-12 + lipid concentrate + apo-transferrin+ monothioglycerol +BSA + insulin + p/s)+ SB431542 (TGFβ inhibitor) + CHIR (Wnt Agonist); and (3) Cells cultured with αMEM + 10% FBS	15	iMSC differentiation through neural crest lineage	80
Jeong et al[46], 20141	NA	(1) iPSCs cultured in iMSC-inducing medium: DMEM/F12 + 20% KOSR + SB431542 (TGFβ inhibitor); and (2) EB grown on matrigel + DMEM/F12 + 0.5% BSA + 10% ITS + SB431542. 3. Outgrowth grown with DMEM/F12 + 10% FBS + p/s	17	Duchene muscular dystrophy	17
Zhao et al[67], 2015	Blood cells	(1) iPSC cultured with mTeSR1 + SB431542 (TGFβ inhibitor) on matrigel-coated plates (7.5% CO2 atmosphere); and (2) Cells cultured with ESC–MSC medium: KO DMEM + KOSR + NEAA + p/s + L-Glutamine + β-ME + bFGF + EGF + SB431542	45	Tumor tropism of iMSC	79

iPSC origin refers to the cell type used for reprograming; Time is indicated as the minimum number of days required to obtain iMSCs; Citations show numbers on March 2020.

¹In references indicate the study includes methods in more than one protocol category. bFGF: Basic fibroblast growth factor; β-ME: β-Mercaptoethanol; BSA: Bovine serum albumin; CARM: Cardiomyogenic medium; CDM: Chemical defined medium; DMEM/F12: Dulbecco's modified Eagle's medium F12; CHIR: Wnt agonist, potent GSK3 inhibitor; EB: Embryoid body; FBS: Fetal bovine serum; FCS: Fetal calf serum; Ham's F12: Medium formulated for single-cell plating of near-diploid Chinese hamster ovary cells; iMSCs: Induced pluripotent stem cell-derived mesenchymal stem cells; iPSC: Induced pluripotent stem cell; ITS: Insulin-transferrin-selenium; KO DMEM: Knockout Dulbecco's modified Eagle's medium; KOSR: Knock-out serum replacement; α-MEM: Minimum essential medium Eagle; MSC: Mesenchymal stem cell; NA: Not available; NEAA: Non-essential amino acid; p/s: Penicillin-streptomycin; TGF: Transforming growth factor.

Table 5 Protocols to produce induced pluripotent stem cell-derived mesenchymal stem cells by approaches that use Platelet Lysate

Ref.	iPSC origin	iPSC to iMSC protocol	Time	Application	Citations
Frobel et al[71], 2014	BM-MSCs	(1) EB formation on ultra-low attachment plates; and (2) Cells cultured with standard medium for MSC: DMEM + L-Glutamine + p/s + hPL + heparin on matrigel-coated wells then passaged on gelatin-coated wells	35	Epigenetic study of iMSC	116
Luzzani et al[72], 2015	Foreskin fibroblasts	(1) iPSC cultured in matrigel/geltrex-coated dishes with a-MEM + 10% PL + p/s + B7 or DMEM + 10% FBS; and (2) Cells cultured with no-coated dishes	20	MSC differentiation using platelet lysate	26
McGrath et al[54], 20191	Dermal fibroblasts	(1) iPSC-MP thawed and expanded in KO DMEM + bFGF + L-Glutamine + MEM NEAA + FBS (20%) + Antibiotic-Antimycotic+ β-ME; and (2) Cell are plated on gelatin coated-plates with DMEM KO + heparin + hPL+ bFGF + L-Glutamine + MEM NEAA + Antibiotic-Antimycotic+ β-ME	NA	iMSC differentiation: GMP-compatible and xeno-free cultivation	6

iPSC origin refers to the cell type used for reprograming; Time is indicated as the minimum number of days required to obtain iMSCs; Citations show numbers on March 2020.

¹In references indicate the study includes methods in more than one protocol category. bFGF: Basic fibroblast growth factor; BM: Bone marrow; β-ME: β-Mercaptoethanol; EB: Embryoid body; DMEM: Dulbecco's modified Eagle's medium; FBS: Fetal bovine serum; GMP: Good manufacturing procedures; hPL: human platelet lysate; hEGF: Human epidermal growth factor; iMSCs: Induced pluripotent stem cell-derived mesenchymal stem cells; iPSC: Induced pluripotent stem cell; KO DMEM: Knockout Dulbecco's modified Eagle's medium; α-MEM: Minimum essential medium Eagle; MSC: Mesenchymal stem cell; NA: Not available; NEAA: Non-essential amino acid; p/s: Penicillin-streptomycin.

Table 6 Frequency of use of iMSC surface markers

Positive CSM	%	Negative CSM	%
CD73	18.1	CD45	24.5
CD105	17.1	CD34	23.0
CD90	15.7	CD14	8.6
CD44	12.5	CD31	7.2
CD29	9.3	HLA-DR	5.8
CD166	6.9	CD11b	5.0
CD146	3.7	CD133	2.9
CD49(a)	2.8	TRA181	2.9
HLA-ABC	2.3	CD19	2.2
CD49(e)	1.9	CD24	2.2
CD106	1.4	CD3	1.4
CD271	1.4	CD40	1.4
CD49(d)	0.9	CD56	1.4
CD140alpha	0.9	CD80	1.4
Sca1	0.9	CD86	1.4
CD33	0.5	Oct3/4	1.4
CD49(f)	0.5	CD4	0.7
CD54	0.5	CD20	0.7
CD71	0.5	CD79a	0.7
CD140(b)	0.5	CD117	0.7
CD144	0.5	CD309	0.7
CD172alpha	0.5	Sox2	0.7
αSMA+	0.5	TRA-160	0.7
Stro1	0.5	TRA-161	0.7
		TRA180	0.7
		SSEA-4	0.7

Positive and negative surface expression markers found in mesenchymal stem cells are listed together with the frequency of findings (%) in the reviewed studies (n = 44). CD: Cluster of differentiation; CSM: Cell surface marker; HLA: Human leukocyte antigen; SSEA: Stage specific embryonic antigen; TRA: Teratocarcinoma surface antigen.