. 2021 Jan 13;11:1101. doi: 10.1038/s41598-020-79651-x

Table 1.

A comparison of popular retinal differentiation protocols against our MG/FF combination protocol, showing the expression timeline of specific cell markers used to confirm the generation of general retinal organoids, retinal ganglion cells (RGCs), retinal pigmented epithelium (RPE) and photoreceptors (PR).

Cell focus	Method			Method adapted from	IHC confirmation		PCR Confirmation		References	Year
Cell focus	Stage	Culture	Days	Method adapted from	Marker	Day	Marker	Day	References	Year
General	SFEBq			Nakano et al.	PAX6	24			⁵⁰	2015
	Embryoid bodies	Floating	0–18		CHX10	24
	Neurospheres	Floating	18+		MITF	24 (low)
					PKCa	24
					BRN3	50
					Crx	50
					NRL	150
General	Embryoid bodies	Floating	0-6	Keller et al.	PAX6	10	PAX6	6	⁵⁴	2009
	Rosettes	Adherent	6–16		RAX	10	RAX	6
	Neurospheres	Floating	16+		CHX10	30	MITF	16
					MITF	30	CHX10	23
					ZO-1	30	RPE65	40
							CRX	60
							OPSIN	70
General	Embryoid bodies	Floating	0-6	Meyer et al.	PAX6	20	PAX6	20	⁵⁷	2011
	Rosettes	Adherent	6-16		CHX10	20–25	BIII	20
	Neurospheres	Floating	16+		ISL1	20–25	BRN3	30
	Seperation of Retinal spheres	Floating	20-25		ZO-1	40	CRX	30
					MITF	40	MITF	40
					BRN3	80	RPE65	40
					PKCa	80	BEST1	40
					CRX	80	NRL	60
					RCVRN	80
					NRL	80
General	SFEBq				PAX6	20	PAX6	10	⁴⁶	2009
	Embryoid Bodies	Floating	0-21		RAX	20	MITF	10
	Neurospheres	Floating	21+		CHX10	20	RAX	20
	PRs	Adherent	90		MITF	35	CHX10	20
					ZO-1	100	RPE65	120
					RHO	140
General	Embryoid Bodies	Adherent	0-28		PAX6	14	LHX2	14	⁴⁸	2017
	Neurospheres	Floating	28+		RAX	14	MITF	14
					LHX2	14	RAX	14
					MITF	14	PAX6	14
					CHX10	14	VSX2	14
					BRN3	21	CRX	14
					CRX	21	BRN3	21
					RCVRN	42	RCVRN	35
General	SFEBq			Nakano et al.	RAX	7	VSX2	10	⁵⁸	2016
	Embryoid Bodies	Floating	0-18		PAX6	7	RAX	10
	Neurospheres	Floating	18-41		LHX2	7	ISL1	10
					MITF	10	ATOH7	10
					VSX2	10	BRN3	10
					BRN3	41	RCVRN	10
							CRX	15
							PAX6	15
							NRL	18
General	Embryoid Bodies	Matrigel	0-4	Ohlemacher et al. and Zhu et al.	RAX	4			This study	2019
	Rosettes	Adherent	4-11		PAX6	4
	Neurospheres	Floating	11+		SOX2	4
					ISL1	28
					HuC/D	28
					PKCa	28
					ZO-1	56
					RCVRN	91
					GFAP	96
					RBPMS	96
					RHO	168
General	Embryoid Bodies	Floating	0-7	Meyer et al.	PAX6	8	PAX6	4	²⁷	2014
	Rosettes	Adherent	7-28		RAX	12	LHX2	4
	Neurospheres	Floating	28+		MITF	14	RAX	8
					VSX2	14
					HuC/D	35
					BRN3	35
					RCVRN	63
					RHO	119
General/PR	SFEBq				PAX6	18			¹⁸	2012
	Embryoid Bodies	Floating	0-18		CHX10	18
	Neurospheres	Floating	18-126		MITF	18
					PKCa	18
					BRN3	30
					RCVRN	43
					NRL	126
					RHO	126
RGC	Embryoid Bodies	Floating	0-4		HuC/D	25	RAX	3	⁶⁰	2016
	RGCs	Adherent	4-30		BIII	25	ISL1	3
					BRN3a	45	ATOH7	3
							PAX6	3
							BRN3B	3
							MITF	8
							RPE65	11
RGC	Embryoid Bodies	Floating	0-4		MATH5	20	RAX	4	²⁸	2018
	Rosettes	Adherent	4-8		BRN3B	40	LHX2	4
	RGCs	Adherent	8-40		ISL1	40	CHX10	12
							MATH5	12
							CRX	12 (low)
							MITF	12
							BRN3B	19
RGC	Rosettes	Adherent	0-14		RAX	14			⁵¹	2014
	Neurospheres	Floating	14-40		PAX6	14
					ZO-1	14
					BRN3	40
RGC	SFEBq			Nakano et al.	RAX	24	RAX	6	⁴⁹	2015
	Neurospheres	Floating	0-26/29		PAX6	24	PAX6	6
	RGCs	Adherent	26/29+		MATH5	34	CRX	6
					BRN3	34	CHX10	6
					CRX	34	PKCa	18
							MITF	18
							BRN3	24
							MATH5	24
RGC/RPE	Embryoid bodies	Floating	0–7	Meyer et al.	PAX6	20	PAX6	20	¹⁹	2015
	Rosettes	Adherent	7-16		CHX10	30	RAX	20
	Neurospheres	Floating	16+		BRN3	40	CHX10	30
	Seperation of Retinal spheres	Floating	20-25		RCVRN	50	BRN3	40
					HuC/D	70	ISL1	50
					ISL1	70	HuC/D	50
		Floating			RBPMS	70
RPE	Embryoid bodies	Floating	0–7		Pax6	13			⁵³	2014
	Rosettes	Adherent	7–16		MITF	13
	Neurospheres	Floating	16+		VSX2	15
					ZO-1	60
RPE	Embryoid Bodies	Matrigel	0-5		PAX6	3	PAX6	5	²⁰	2013
	RPE	Adherent	5-25		ZO-1	1 (EB)	RAX	5
					RAX	5	CHX10	5
					CHX10	15	MITF	10
PR/RPE	Embryoid Bodies	Floating	0-30		Rho	45	PAX6	15	⁴⁷	2012
	Neurospheres	Adherent	30-60		OPN1SW	45	RPE65	30
					RPE65	45	CHX10	45
					ZO-1	45	CRX	60
							RCVRN	60
PR	SFEBq			Nakano et al.	PAX6	37			⁵⁶	2015
	Embryoid Bodies	Floating	0-12		BRN3b	37
	Neurospheres	Floating	12-90		CRX	37
					RCVRN	37
					PKCa	67
					Arr	67
					NRL	67
					OPN1SW	90
PR	Embryoid Bodies	Floating	0-6	Meyer et al.	PAX6	56	RAX	56	⁵⁵	2018
	Neurospheres	Adherent	6+		RAX	56	VSX2	56
					RCVRN	56	PAX6	56
					CRX	56	RCVRN	56
					BRN3B	140	CRX	56
							MITF	56
PR	SFEBq			Nakano et al.	BRN3	35	OPN1SW	12	³⁶	2017
	Embryoid Bodies	Floating	0-10/12		RCVRN	45	VSX2	30
	Neurospheres	Floating	10/12+		RHO	160	RCVRN	30
							CRX	65
							NRL	100
							ARR3	100