PTF1a Activity in Enriched Posterior Foregut Endoderm, but Not Definitive Endoderm, Leads to Enhanced Pancreatic Differentiation in an In Vitro Mouse ESC-Based Model

Table 1

Summary of the experimental procedure and results of several endoderm differentiation protocols that were tested on the Tet-Ptf1a cells indicating the degree of differentiation to definitive endoderm, Pdx1⁺ cells, and Nkx6.1⁺ cells.

Protocol	Media	Growth factor conc.	Cell seeding number/culture format	Sox17^+Foxa2^+ cells	PTF1a induced/days	Pdx1^+ cells	Pdx1^+ cells on prolonged culture
EB in 15% FBS [24]	15% FBS, DMEM for 7 days + 1% SR, DMEM for 2 days	None	3 × 10^6 cells in 60 mm dish	10%- EB7+2; 25%- EB7+4; Not all EBs have copositive cells	Yes. Sequential/3 d	Begin to see at EB7+7	Yes
Bernardo protocol in monolayer [28]	DMEM (Iscove’s modified DM plus Ham’s F12 medium at a 1 : 1 ratio), L-Glut, BSA 5 g/L, lipids 1x, 2x ITS, BME 1x.	A100B10 for 3 days; A100B10 for 3 days + A100 for 2 days	500,000 per 24 well	Very few and cells unhealthy; 5% copositive cells around the edges of colonies	Yes. Sequential/3 days	None; 2 clusters (50 cells each)	None; None
	0.5% serum, DMEM, 1% L-glut, 1% penstrep	A100B10 for 3 days		Almost none		None	None
Low serum		A100B10 for 3 days + A100 for 2 days	500,000 per 24 well	Few cells but culture looks unhealthy	Yes. Sequential/3 days	None	None
	2% serum, DMEM, 1% L-glut, 1% penstrep	A100B10 for 3 days		Few cells and culture looks little better		None	None
		A100B10 for 3 days + A100 for 2 days		5–8% copositive cells where density was low		None	None
Xu protocol w/o Matrigel [17]	DMEM high glucose, 2 g/L BSA, 1% penstrep, 1% L-Glut	A100F100B10	300,000 per 24 well	2-3%	No		None
		A100F100B50		1-2%	No		None
		A100B10		1-2%	No		None
		A100B50		<1%	No		None
		A100F100B10	100,000 per 24 well	10%	No		None
		A100F100B50		3-4%	No		None
		A100B100		10%	No
		A100B50		5%	No
Hansson protocol [5]	KO DMEM + 1x-N2 1x-B27 + 1% L-glut + 1% penstrep + Bme + NEAA for 5 days	A100	1000 per 96 well	50–60%	Yes, (a) overlapping (b) sequential/3 d	Yes, (a) scattered cells (b) scattered	Did not see Pdx1^+ cells on prolonged culture
		A30		40–50%		Yes, scattered cells
		A100	5000 per 96 well	10%	No
		A30		10%	No
Melton protocol in 96 well [7]	ARPMI + 0.2% FBS + 1% L-glut-penstep	Nodal1000	1000 per 96 well	>90%	Yes, (a) overlapping (b) sequential/3 d	(a) No (b) 15 cell clusters with 20–100 cells each
		IDE 800 nM		30%		(a) and (b) No
		IDE 5 μM		High 40–50%	No
		Nodal1000	5000 per 96 well	>80%	Yes, (a) overlapping (b) sequential/3 d	(a) Scattered cells (b) No
		IDE 800 nM		>60%		(a) No (b) Scattered cells
		IDE 5 μM		40–50%	No
Melton protocol on 24 well	ARPMI + 0.2% FBS + 1% L-glut-penstep	IDE 800 nM	7500 per 24 well	10%	Yes/3 d	None
			15000 per 24 well	5%	Yes/3 d	None
		IDE 800 nM		25%	No
Melton protocol on 24 well w Matrigel	ARPMI + 0.2% FBS + 1% L-glut-penstep on Matrigel	Nodal1000	7500	25%	No
		A100		1%	No
Melton modified (obtained from [7])	Definitive endoderm: Activin A (50 ng/mL) or Nodal (1000 ng/mL) in 0.2% FBS in ARPMI for 4–6 days; Primitive gut tube: FGF10 (50 ng/mL), KAAD-cyc (0.25 μM) in 2% FBS in ARPMI for 2 days; Pancreatic endoderm: FGF10 (50 ng/mL), KAAD-cyc (0.25 μM), RA (2 μM) in B27 DMEM for 4 days; PTF1a induction for 4 days in B27 DMEM or 1% SR for; Additional culture for 4 more days in same media		2000 per 96 well fed every other day	Activin ~70%. Nodal: 80–90%.; After treatment with further factors, some Pdx1 seen in nodal cultures before PTF1a induction	Yes.; Sequential/4 d	Activin A: Yes, scattered Pdx1^+ cells; Nodal-yes, Pdx1 in bud-like clusters.	Did not see Pdx1 on culturing for 4 more days	Nkx6.1 expression was seen in some of the Pdx1^+ cells but these cells did not become insulin^+ even on ra + nic treatment.