Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Summary

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Abstract

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

Introduction

The transparent cornea at the ocular surface allows light to enter the retina and provides the majority of the eye's refractive power. The outermost layer, the stratified corneal epithelium, is continuously regenerated by limbal epithelial stem cells (LESCs). The LESCs reside in the basal layer of the limbal niches at the corneoscleral junction^1,2. LESCs lack specific and unique markers, so their identification requires a more extensive analysis of a set of putative markers. Epithelial transcription factor p63, and especially N-terminally truncated transcript of the alpha isoform of p63 (ΔNp63α),....

Protocol

University of Tampere has the approval of the National Authority for Medicolegal affairs Finland (Dnro 1426/32/300/05) to conduct research on human embryos. The institute also has supportive statements of the Ethical Committee of the Pirkanmaa Hospital District to derive, culture, and differentiate hESC lines (Skottman/R05116) and to use hiPSC lines in ophthalmic research (Skottman/R14023). No new cell lines were derived for this study.

NOTE: The protocol described is based on specific, commercially available hPSC and corneal epithelium differentiation media. Please refer to the Table of Materials for manufacturer/supplier ....

Results

From hPSCs to hPSC-LESCs

The entire process from inducing differentiation of FF hPSCs to cryostoring hPSC-LESCs takes around 3.5 weeks. Schematic overview of the differentiation method highlighting its key steps is presented in Figure 1A. Figure 1B shows typical morphologies of cell populations in different phases of the protocol. The data presented are obtained with Regea.......

Discussion

The expected result of this protocol is the successful and robust generation of LESCs from a single cell suspension of FF hPSC within approximately 3.5 weeks. As corneal epithelium develops from surface ectoderm²⁹, the first step of the protocol aims at steering hPSCs towards this lineage. A short 24 h induction with transforming growth factor beta (TGF-β) antagonist SB-505124, and bFGF are used to induce ectodermal differentiation, followed by 48 h mesodermal BMP-4 cue to push the cells towa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Material/Reagent
1x DPBS containing Ca2+ and Mg2+	Gibco	#14040-091
1x DPBS without Ca2+ and Mg2+	Lonza	#17-512F/12
100 mm cell culture dish	Corning CellBIND	#3296	Culture vessel format for adherent hPSC-LESC differentiation
12-well plate	Corning CellBIND	#3336	Culture vessel format for IF samples
24-well plate	Corning CellBIND	#3337	Culture vessel format for IF samples
2-mercaptoethanol	Gibco	#31350-010
6-well plate, Ultra-Low attachment	Corning Costar	#3471	Culture vessel format for induction in suspension culture
Alexa Fluor 488 anti-mouse Ig	ThermoFisher Scientific	#A-21202	Secondary antibody for IF
Alexa Fluor 488 anti-rabbit Ig	ThermoFisher Scientific	#A-21206	Secondary antibody for IF
Alexa Fluor 568 anti-goat Ig	ThermoFisher Scientific	#A-11057	Secondary antibody for IF
Alexa Fluor 568 anti-mouse Ig	ThermoFisher Scientific	#A-10037	Secondary antibody for IF
Basic fibroblast growth factor (bFGF, human)	PeproTech Inc.	#AF-100-18B	Animal-Free Recombinant Human FGF-basic (154 a.a.)
BD Cytofix/Cytoperm Fixation/Permeabilization Solution	BD Biosciences	#554722	Fixation and permeabilization solution for flow cytometry
BD Perm/Wash Buffer	BD Biosciences	#554723	Washing buffer for flow cytometry
Blebbistatin	Sigma-Aldrich	#B0560
Bone morphogenetic protein 4 (BMP-4)	PeproTech Inc.	#120-05A
Bovine serum albumin (BSA)	Sigma-Aldrich	#A8022-100G
Cytokeratin 12 antibody	Santa Cruz Biotechnology	#SC-17099	Primary antibody for IF
Cytokeratin 14 antibody	R&D Systems	#MAB3164	Primary antibody for IF
Cytokeratin 15 antibody	ThermoFisher Scientific	#MS-1068-P	Primary antibody for IF
CnT-30	CELLnTEC Advanced Cell Systems AG	#Cnt-30	Culture medium for adherent hPSC-LESC differentiation
Collagen type IV (human)	Sigma-Aldrich	#C5533	Human placental collagen type IV
CoolCell LX Freezing Container	Sigma-Aldrich	#BCS-405
CryoPure tubes	Sarsted	#72.380	1.6 mL cryotube for hPSC-LESC cryopreservation
Defined Trypsin Inhibitor	Gibco	#R-007-100
Essential 8 Flex Medium Kit	Thermo Fisher Scientific	#A2858501
GlutaMAX	Gibco	#35050061
Laminin 521	Biolamina	#Ln521	Human recombinant laminin 521
ΔNp63α antibody	BioCare Medical	#4892	Primary antibody for IF
OCT3/4 antibody	R&D Systems	#AF1759	Primary antibody for IF
p63α antibody	Cell Signaling Technology	#ACI3066A	Primary antibody for IF
p63-α (D2K8X) XP Rabbit mAb (PE Conjugate)	Cell Signaling Technology	#56687	p63-α PE-conjugated antibody for flow cytometry
PAX6 antibody	Sigma-Aldrich	#HPA030775	Primary antibody for IF
Penicillin/Streptomycin	Lonza	#17-602E
Paraformaldehyde (PFA)	Sigma-Aldrich	#158127	Cell fixative for IF
ProLong Gold Antifade Mountant with DAPI	Thermo Fisher Scientific	#P36931	DAPI mountant for hard mounting for IF
PSC Cryopreservation Kit	Thermo Fisher Scientific	#A2644601
TrypLE Select Enzyme	Gibco	#12563-011
KnockOut Dulbecco’s modified Eagle’s medium	Gibco	#10829018
KnockOut SR XenoFree CTS	Gibco	#10828028
MEM non-essential amino acids	Gibco	#11140050
SB-505124	Sigma-Aldrich	#S4696
Triton X-100	Sigma-Aldrich	#T8787	Permeabilization agent for IF
VectaShield	Vector Laboratories	#H-1200	DAPI mountant for liquid mounting for IF
Name	Company	Catalog Number	Comments
Equipment
Cytocentrifuge, e.g. CellSpin II	Tharmac
Flow cytometer, e.g. BD Accuri C6	BD Biosciences
Fluorescence microscope, e.g.Olympus IX 51	Olympus