Perspectives of pluripotent stem cells in livestock

Table 1 Most recent examples of embryonic stem cells successfully generated from livestock

Species	Embryonic stage	Culture medium and condition	Expression of pluripotency markers	Long-term culture (passage)	Karyotype	In vitro differentiation to EBs	In vivo differentiation to teratoma	Germ line transmission	Ref.
Cattle	CDX2-KD blastocysts	KO-DMEM, 2 mM glutamine, 1% MEM-NEAAs, 20 ng/mL hrFGF, 20 ng/mL hrLIF, 0.1 mM β–mercaptoethanol, 15% FBS + MEF under 37℃, 5% CO2	Yes	37	Normal	Yes	Yes	No	[272]
	Blastocysts	CTFR medium contains low fatty acid BSA, 20 ng/mL hFGF2, 2.5 μM IWR1 + MEFs under 37℃, 5% CO2	Yes	> 70	Normal	No	Yes	No	[145]
Buffalo	Blastocysts	KO-DMEM, 15% KSR, 2 mM L-glutamine, 50 μg/mL gentamicin sulfate, 1% MEM-NEAAs, 0.1 mM β-mercaptoethanol, 1000 IU/mL mLIF, 5 ng/mL FGF2 + BFF under 37℃, 5% CO2	Yes	135	Normal	Yes	No	No	[20]
	Blastocysts	DMEM, 20% FBS, 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, 2% NEAA, 1% ITS, 50 μg/mL gentamycin sulfate, 30 ng/mL LIF, 40 ng/mL bFGF + BFF under 38.5℃, 5% CO2	Yes	15	Normal	No	Yes	No	[273]
Ovine	Blastocysts	DMEM high glucose, 2 mM L-glutamine, 1 mM Na-Pyruvate, 0.1 mM β-mercaptoethanol, 0.1 mM NEAAs, 10 ng/mL LIF, 20 mg/mL insulin, 1000 IU/mL penicillin, 10 mg/mL streptomycin + STO under 38.5℃, 5% CO2	Yes	No	Normal	Yes	No	No	[274]
	Blastocysts	DMEM/F12 supplemented with N2, B27, GSK3 inhibitor (CHIR99021), rhbFGF + OEF or MEF under 38.5℃, 5% CO2	Yes	30	No	Yes	Yes	No	[275]
Caprine	Blastocysts	DMEM, 20% FCS, 1000 IU/mL mLIF, 1% NEAAs 0.1 mM β-mercaptoethanol, 2 mM l-glutamine + GFF under 38.5℃, 5% CO2	Yes	15	Normal	Yes	No	No	[17]
	Blastocysts	DMEM, 0.1 mM 2-mercaptoethanol, 0.1 mM MEM-NEAAs, 2 mM L-glutamine, 10% FBS, 1000 U/mL hLIF + GFF under 37℃, 5% CO2	Yes	120	Normal	Yes	Yes	No	[16]
Porcine	Blastocysts	1:1 ratio of 1. α-MEM medium supplemented with 10% KSR, 0.05 mM β-mercaptoethanol, 1% NEAAs, 1% antibiotic-antimycotic, 4 ng/mL EGF, 10 μL/mL 100 × ITS, 1000 U/mL mLIF, 2 ng/mL bFGF and, 2. DMEM/F-10-based medium supplemented with 15% heat-inactivated FBS, 0.2 mM β-mercaptoethanol, 1% NEAA, 1% antibiotic–antimycotic and 2 ng/mL bFGF + MEFs under 37℃, 5% CO2	Yes	19	Normal	Yes	No	No	[276]
	Blastocysts	α-MEM, 20% KSR, 20 ng/mL bFGF, 20 ng/mL EGF, 10 ng/mL Activin-a, 1% ITS, 1 mM MEM-NEAAs, 55 μM β2-mercaptoethanol + STO at 38.5℃, 5% CO2	Yes	21	Normal	Yes	Yes	No	[277]
	Blastocysts	DMEM, 20% KSR and N2B27 medium, 1% NEAAs, 2 mM L-glutamine, 1% PS, 0.1 mM b-mercaptoethanol, 3 mM CHIR99021, 1 mM PD0325901, 2 mM SB, and 50 ng/mL vitamin C + MEFs under 38.5℃, 5% CO2	Yes	139	Normal	Yes	Yes	No	[278]
Equine	Blastocysts	DMEM/F12, 15% FCS, 1000 U/mL hLIF, 15% FBS + MEF under 38.5℃, 5% CO2	Yes	28	Normal	Yes	No	No	[279]
	Blastocysts	KO-DMEM, 15% FBS, 0.1 mM NEAAs, 2 mM L-glutamine, 1% ITS, 100 μg/mL streptomycin, 100 IU/mL penicillin, 0.1 mM β-mercaptoethanol, hLIF, hbFGF + MEF under 38.5℃, 5% CO2	Yes	15	No	No	No	No	[280]
Canine	Blastocysts	KO-DMEM/Ham’s F12, 15% KSR, 1 × GlutaMAX, 1 × NEAAs, R3IGF1, 0.1 mM 2-mercaptoethanol, 10 ng/mL hrLIF, 4 ng/mL rhFGF2, 0.5 μM, MEK inhibitor PD0325901, 3 μM GSK3β inhibitor CHIR99021 + MEFs under 37℃, 5% CO2	Yes	--	Normal	Yes	Yes	No	[281]
	Blastocysts	KO-DMEM or DMEM/-12, 0.1 mM β-mercaptoethanol, 5 μM thymidine, 15 μM cytidine, 15 μM guanosine, 15 μM adenosine and 15 μM uridine nucleosides, 0.2 mM GlutaMax, 0.1 mM NEAAs, penicillin (100 IU/mL), streptomycin (50 μg/mL), 10 ng/mL hLIF, 4 ng/mL hbFGF, 15% FBS or KSR + MEFs under 37.5℃, 5% CO2	Yes	30	Normal	Yes	Yes	No	[99]

CDX2-KD: CDX2 gene knockdown; KO-DMEM: knockout Dulbecco's modified Eagle's medium; DMEM: Dulbecco's modified Eagle's medium; MEM-NEAA: Minimum Essential Medium-non-essential amino acids; hrFGF: Human recombinant fibroblast growth factor; hrLIF: Human recombinant leukemia inhibitory factor; hFGF: Human fibroblast growth factor; DMEM/F12: Dulbecco's modified Eagle's medium/nutrient mixture F-12;‎ FBS: Fetal bovine serum; MEF: Mouse embryonic fibroblast; mLIF: Mouse leukemia inhibitory factor; FGF2: Fibroblast growth factor 2; ITS: Insulin–transferrin–selenium; LIF: Leukemia inhibitory factor; bFGF: Basic growth factor; rhbFGF: Recombinant human basic fibroblast growth factor; OEF: Ovine embryonic fibroblast; GFF: Goat fetal fibroblast; hLIF: Human leukemia inhibitory factor; PS: Penicillin-streptomycin; BSA: Bovine serum albumin; FCS: Fetal calf serum; KSR: Knockout serum replacer; EGF: Epidermal growth factor; BFF: Buffalo fetal fibroblast; STO: Sandos inbred mouse-derived 6-thioguanine-and ouabain-resistant; SB: SB431542 inhibitor.

Table 2 Most recent examples of induced pluripotent stem cells successfully generated from livestock

Species	Cell type	Culture medium and condition	Expression of pluripotency markers	Long-term culture (passage)	Karyotype	In vitro differentiation to EBs	In vivo differentiation to teratomas	Germline transmission	Ref.
Cattle	Fetal fibroblasts	DMEM/F-12, 20% KSR, 1 mM l-glutamine, 0.1 mM NEAAs, 0.1 mM mercaptoethanol, 100 U/mL penicillin, 100 μg/mL streptomycin, 8 ng/mL bFGF, 1000 U/mL hLIF on MEF at 37℃ and 5% CO2	Yes	40	Normal	Yes	Yes	No	[58]
	Fetal fibroblasts	KO-DMEM, 15% FBS, 2 mM L-glutamine, 1% NEAAs, 0.1 mM β-mercaptoethanol, 106 U/mL hLIF, 10 ng/mL bFGF on STO at 37℃ and 5% CO2	Yes	50	Normal	Yes	Yes	No	[130]
Buffalo	Fetal fibroblasts	DMEM high glucose, 20% ESC-FBS, 2 mM l-glutamine, 1% NEAAs, 0.1 mM β-mercaptoethanol, 10 ng/mL bFGF, 10 ng/mL LIF on MEF at 37℃ and 5% CO2	Yes	10	Normal	Yes	Yes	No	[65]
	Fetal fibroblasts	DMEM/F-12, 20% KSR, 0.1 mM NEAAs, 1 mM L-glutamine, 0.1 mM mercaptoethanol, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 ng/mL bFGF, 1000 U/mL hLIF on gelatine at 37℃ and 5% CO2	Yes	15	Normal	Yes	No	No	[66]
Ovine	Embryonic fibroblasts	DMEM, 20% FBS, 1% ITS, 0.1 mM 2-β mercaptoethanol, 1 mM NEAAs, 2 mM glutamine, 4 ng/mL bFGF, 1000 U/mL mLIF on MEFs at 37℃ and 5% CO2	Yes	17	Normal	Yes	Yes	Yes (formation of ICM in tetraploid)	[50]
	Embryonic fibroblasts	KO-DMEM, KSR, 0.1 mM NEAAs, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 8 ng/mL hFGF2, 1000 U/mL mLIF on SNL at 37℃ and 5% CO2	Yes	23	Normal	Yes	Yes	Yes (live-born chimeric lambs)	[67]
Caprine	Fetal fibroblasts	DMEM/F12, 20% KSR, 1 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 1% NEAAs, 2% sodium bicarbonate solution, 1000 IU/mL 2i/LIF, 4 ng/mL bFGF on STO at 37℃ and 5% CO2	Yes	30	Normal	Yes	Yes	No	[282]
	Embryonic fibroblasts	KO-DMEM, 20% KSR, 1% NEAA, 1% L-glutamine, 0.1 mM EAA, 1% penicillin /streptomycin, 10 ng/mL FGF2 on GEF at 37℃ and 5% CO2	Yes	22	Normal	Yes	No	No	[283]
Porcine	Embryonic fibroblasts and microvascular pericyte cells	LCDMV medium contains 50% neurobasal medium, 50% DMEM/F12, 1 × N2, 0.5 × B27, 5% KSR, 10 ng/mL LIF, 1 μM CHIR99021, 2 μM (S)-(+)-dimethindene maleate, 2 μM minocycline hydrochloride, 40 μg/mL vitamin C on MEF at 37℃ and 5% CO2	Yes	28	Normal	Yes	Yes	Yes (chimeric formation in post-implantation pig conceptuses)	[284]
	Sertoli cells	DMEM/F12, 10% KSR, 10% FBS, 1 mM l-glutamine, 1 mM antibiotic, 1% NEAAs, 0.1 mM β-mercaptoethanol, 10 ng/mL bFGF, 10 ng/mL hLIF on MEF at 37℃ and 5% CO2	Yes	50	Normal	Yes	Yes	No	[285]
Equine	Fetal fibroblasts	DMEM/F12, 20% KSR, 10 ng/mL bFGF, 1% penicillin/streptomycin, 10 ng/mL hLIF on MEF at 37℃ and 5% CO2	Yes	25	--	Yes	No	No	[286]
	Fetal fibroblasts	DMEM, 20% FBS or KO-DMEM, 20% KSR, 2 mM l-glutamine, 0.1 mM β-mercaptoethanol, 0.1 mM MEM- NEAAs, 1% penicillin–streptomycin, 8 ng/mL hbFGF, 1000 U/mL hLIF on SNL at 37℃ and 5% CO2	Yes	30	Normal	Yes	Yes	No	[61]
Canine	Embryonic fibroblasts	Serum-free N2B27-based medium, 4 ng/mL hbFGF on MEF at 37℃ and 5% CO2	Yes	50	Normal	Yes	No	No	[287]
	Fetal fibroblasts	KO-DMEM/F12, 20% KSR, 2 mM L-glutamine, 0.1 mM NEAAs, 0.1 mM β-mercaptoethanol, 0.1 mM bFGF on MEF at 37℃ and 5% CO2	Yes	15	Normal	Yes	Yes	No	[288]

DMEM: Dulbecco's modified Eagle's medium; KO-DMEM: Knockout Dulbecco's modified Eagle's medium; DMEM/F12: Dulbecco's modified Eagle's medium/nutrient mixture F-12;‎ NEAAs: Non-essential amino acids; bFGF: Basic fibroblast growth factor; hLIF: Human leukemia inhibitory factor; MEF: Mouse embryonic fibroblast; KSR: Knock-out serum replacement; FBS: Fetal bovine serum; LIF: Leukemia inhibitory factor; ESC-FBS: Embryonic stem cells-fetal bovine serum; ITS: Insulin–transferrin–selenium; mLIF: Mouse leukemia inhibitory factor; hFGF2: Human fibroblast growth factor 2; EAAs: Essential amino acids; hbFGF: Human basic fibroblast growth factor; STO: Sandos inbred mouse-derived 6-thioguanine-and ouabain-resistant.