The study was supported by the Academy of Finland (grant number 297886), the Human spare parts program of Tekes, the Finnish Funding Agency for Technology and Innovation, the Finnish Eye and Tissue Bank Foundation and the Finnish Cultural Foundation. The authors thank the biomedical laboratory technicians Outi Melin, Hanna Pekkanen, Emma Vikstedt, and Outi Heikkil&#228; for excellent technical assistance and contribution to cell culture. Professor Katriina Aalto-Set&#228;l&#228; is acknowledged for providing the hiPSC line used and BioMediTech Imaging Core facility for providing equipment for fluorescence imaging.

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, commer...

<ol>
	<li>Dua, H. S., Shanmuganathan, V. A., Powell-Richards, A. O., Tighe, P. J., Joseph, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limbal+epithelial+crypts:+a+novel+anatomical+structure+and+a+putative+limbal+stem+cell+niche.">Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche.</a> The British Journal of Ophthalmology. 89 (5), 529-532 (2005).</li><li>Yazdanpanah, G., Jabbehdari, S., Djalilian, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limbal+and+corneal+epithelial+homeostasis.">Limbal and corneal epithelial homeostasis.</a> Current Opinion in Ophthalmology. 28 (4), 348-354 (2017).</li><li>Di Iorio, E., Barbaro, V., Ruzza, A., Ponzin, D., Pellegrini, G., De Luca, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoforms+of+DeltaNp63+and+the+migration+of+ocular+limbal+cells+in+human+corneal+regeneration.">Isoforms of DeltaNp63 and the migration of ocular limbal cells in human corneal regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 9523-9528 (2005).</li><li>Rama, P., Matuska, S., Paganoni, G., Spinelli, A., De Luca, M., Pellegrini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limbal+stem-cell+therapy+and+long-term+corneal+regeneration.">Limbal stem-cell therapy and long-term corneal regeneration.</a> The New England Journal of Medicine. 363 (2), 147-155 (2010).</li><li>Notara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+sickness+and+in+health:+Corneal+epithelial+stem+cell+biology,+pathology+and+therapy.">In sickness and in health: Corneal epithelial stem cell biology, pathology and therapy.</a> Experimental Eye Research. 90 (2), 188-195 (2010).</li><li>Osei-Bempong, C., Figueiredo, F. C., Lako, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+limbal+epithelium+of+the+eye--a+review+of+limbal+stem+cell+biology,+disease+and+treatment.">The limbal epithelium of the eye--a review of limbal stem cell biology, disease and treatment.</a> BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology. 35 (3), 211-219 (2013).</li><li>Kolli, S., Ahmad, S., Lako, M., Figueiredo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+clinical+implementation+of+corneal+epithelial+stem+cell+therapy+for+treatment+of+unilateral+limbal+stem+cell+deficiency.">Successful clinical implementation of corneal epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency.</a> Stem Cells. 28 (3), 597-610 (2010).</li><li>Sangwan, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+outcomes+of+xeno-free+autologous+cultivated+limbal+epithelial+transplantation:+a+10-year+study.">Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study.</a> The British Journal of Ophthalmology. 95 (11), 1525-1529 (2011).</li><li>Basu, S., Mohan, S., Bhalekar, S., Singh, V., Sangwan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+limbal+epithelial+transplantation+(SLET)+in+failed+cultivated+limbal+epithelial+transplantation+(CLET)+for+unilateral+chronic+ocular+burns.">Simple limbal epithelial transplantation (SLET) in failed cultivated limbal epithelial transplantation (CLET) for unilateral chronic ocular burns.</a> The British Journal of Ophthalmology. , (2018).</li><li>Ahmad, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+embryonic+stem+cells+into+corneal+epithelial-like+cells+by+in+vitro+replication+of+the+corneal+epithelial+stem+cell+niche.">Differentiation of human embryonic stem cells into corneal epithelial-like cells by in vitro replication of the corneal epithelial stem cell niche.</a> Stem Cells. 25 (5), 1145-1155 (2007).</li><li>Hanson, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+human+embryonic+stem+cells+onto+a+partially+wounded+human+cornea+in+vitro.">Transplantation of human embryonic stem cells onto a partially wounded human cornea in vitro.</a> Acta Ophthalmologica. 91 (2), 127-130 (2013).</li><li>Hayashi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+corneal+epithelial+cells+from+induced+pluripotent+stem+cells+derived+from+human+dermal+fibroblast+and+corneal+limbal+epithelium.">Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.</a> PLoS ONE. 7 (9), e45435 (2012).</li><li>Hewitt, K. 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A., Nieto-Nicolau, N., Morales-Pastor, A., Casaroli-Marano, R. 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R., Sowden, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine:+DIY+eye.">Regenerative medicine: DIY eye.</a> Nature. 472 (7341), 42-43 (2011).</li><li>International Stem Cell Initiative, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+ethnically+diverse+human+embryonic+stem+cells+identifies+a+chromosome+20+minimal+amplicon+conferring+growth+advantage.">Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage.</a> Nature Biotechnology. 29 (12), 1132-1144 (2011).</li><li>Foster, J. W., Wahlin, K., Adams, S. M., Birk, D. E., Zack, D. 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The authors have nothing to disclose.

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-&#946;) antagonist SB-505124, and bFGF are used to induce ectodermal differentiation, followed by 48 h mesodermal BMP-4 cue to push the cells towa...

The transparent cornea at the ocular surface allows light to enter the retina and provides the majority of the eye&#39;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 junction1,2. 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 (&#916;Np63&#945;), has been proposed as a relevant positive LESC marker3,4. Asymmetric division of LESCs allows them to self-renew, but also produce progeny that migrate centripetally and anteriorly. As the cells progress toward the corneal surface they gradually lose their stemness and finally terminally differentiate to superficial squamous cells that are continuously lost from the corneal surface.
Damage to any of the corneal layers can lead to severe visual impairment, and corneal defects are thus one of the leading causes of vision loss worldwide. In limbal stem cell deficiency (LSCD), the limbus is destroyed by disease or trauma which leads to conjunctivalization and opacification of the corneal surface and subsequent loss of vision5,6. Cell replacement therapy using autologous or allogeneic limbal grafts offers a treatment strategy for patients with LSCD4,7,8,9. However, harvesting autologous grafts bears a risk of complications to the healthy eye, and donor tissue is in short supply. Human pluripotent stem cells (hPSCs), specifically human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), can serve as an unlimited source of clinically relevant cell types, including corneal epithelial cells. Therefore, hPSC-derived LESCs (hPSC-LESCs) represent an attractive new cell source for ocular cell replacement therapy.
Traditionally, both the undifferentiated hPSC culture methods and their differentiation protocols to LESCs have relied on the use of undefined feeder cells, animal sera, conditioned media, or amniotic membranes10,11,12,13,14,15. Recently, efforts toward safer cell therapy products have prompted the search for more standardized and xeno-free culture and differentiation protocols. As a result, several defined and xeno-free methods for long-term culture of undifferentiated hPSCs are now commercially available16,17,18. As a continuum, directed differentiation protocols relying on molecular cues to guide hPSCs to corneal epithelial fate have been recently introduced19,20,21,22,23. Yet many of these protocols used either undefined, feeder based hPSCs as starting material, or complex, xenogeneic growth factor cocktails for differentiation.
The purpose of this protocol is to provide a robust, optimized, xeno-and feeder-free hPSC culture method and subsequent differentiation to corneal LESCs. Monolayer culture of pluripotent hPSCs on laminin-521 (LN-521) matrix in defined, albumin-free hPSC medium (specifically Essential 8 Flex) allows rapid production of homogeneous starting material for differentiations. Thereafter a simple, two-step differentiation strategy guides hPSCs toward surface ectodermal fate in suspension, followed by adherent differentiation to LESCs. A cell population where &#62; 65% express &#916;Np63&#945; is obtained within 24 days. The xeno- and feeder-free protocol has been tested with several hPSC lines (both hESCs and hiPSCs), without any requirement for cell line specific optimization. The protocols for weekend-free maintenance, passaging, cryostoring and hPSC-LESC phenotyping described here enable production of large batches of high-quality LESCs for clinical or research purposes.

<table>
	<tbody>
		<tr>
			<td>Material/Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1x DPBS containing Ca2+ and Mg2+</td>
			<td>Gibco</td>
			<td>#14040-091</td>
			<td></td>
		</tr>
		<tr>
			<td>1x DPBS without Ca2+ and Mg2+</td>
			<td>Lonza</td>
			<td>#17-512F/12</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm cell culture dish</td>
			<td>Corning CellBIND</td>
			<td>#3296</td>
			<td>Culture vessel format for adherent hPSC-LESC differentiation</td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Corning CellBIND</td>
			<td>#3336</td>
			<td>Culture vessel format for IF samples</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Corning CellBIND</td>
			<td>#3337</td>
			<td>Culture vessel format for IF samples</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Gibco</td>
			<td>#31350-010</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate, Ultra-Low attachment</td>
			<td>Corning Costar</td>
			<td>#3471</td>
			<td>Culture vessel format for induction in suspension culture</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 anti-mouse Ig</td>
			<td>ThermoFisher Scientific</td>
			<td>#A-21202</td>
			<td>Secondary antibody for IF</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 anti-rabbit Ig</td>
			<td>ThermoFisher Scientific</td>
			<td>#A-21206</td>
			<td>Secondary antibody for IF</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 anti-goat Ig</td>
			<td>ThermoFisher Scientific</td>
			<td>#A-11057</td>
			<td>Secondary antibody for IF</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 anti-mouse Ig</td>
			<td>ThermoFisher Scientific</td>
			<td>#A-10037</td>
			<td>Secondary antibody for IF</td>
		</tr>
		<tr>
			<td>Basic fibroblast growth factor (bFGF, human)</td>
			<td>PeproTech Inc.</td>
			<td>#AF-100-18B</td>
			<td>Animal-Free Recombinant Human FGF-basic (154 a.a.)</td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm Fixation/Permeabilization Solution</td>
			<td>BD Biosciences</td>
			<td>#554722</td>
			<td>Fixation and permeabilization solution for flow cytometry</td>
		</tr>
		<tr>
			<td>BD Perm/Wash Buffer</td>
			<td>BD Biosciences</td>
			<td>#554723</td>
			<td>Washing buffer for flow cytometry</td>
		</tr>
		<tr>
			<td>Blebbistatin</td>
			<td>Sigma-Aldrich</td>
			<td>#B0560</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone morphogenetic protein 4 (BMP-4)</td>
			<td>PeproTech Inc.</td>
			<td>#120-05A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>#A8022-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokeratin 12 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>#SC-17099</td>
			<td>Primary antibody for IF</td>
		</tr>
		<tr>
			<td>Cytokeratin 14 antibody</td>
			<td>R&#38;D Systems</td>
			<td>#MAB3164</td>
			<td>Primary antibody for IF</td>
		</tr>
		<tr>
			<td>Cytokeratin 15 antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>#MS-1068-P</td>
			<td>Primary antibody for IF</td>
		</tr>
		<tr>
			<td>CnT-30</td>
			<td>CELLnTEC Advanced Cell Systems AG</td>
			<td>#Cnt-30</td>
			<td>Culture medium for adherent hPSC-LESC differentiation</td>
		</tr>
		<tr>
			<td>Collagen type IV (human)</td>
			<td>Sigma-Aldrich</td>
			<td>#C5533</td>
			<td>Human placental collagen type IV</td>
		</tr>
		<tr>
			<td>CoolCell LX Freezing Container</td>
			<td>Sigma-Aldrich</td>
			<td>#BCS-405</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoPure tubes</td>
			<td>Sarsted</td>
			<td>#72.380</td>
			<td>1.6 ml cryotube for hPSC-LESC cryopreservation</td>
		</tr>
		<tr>
			<td>Defined Trypsin Inhibitor</td>
			<td>Gibco</td>
			<td>#R-007-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential 8 Flex Medium Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>#A2858501</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>#35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin 521</td>
			<td>Biolamina</td>
			<td>#Ln521</td>
			<td>Human recombinant laminin 521</td>
		</tr>
		<tr>
			<td>&#916;Np63&#945; antibody</td>
			<td>BioCare Medical</td>
			<td>#4892</td>
			<td>Primary antibody for IF</td>
		</tr>
		<tr>
			<td>OCT3/4 antibody</td>
			<td>R&#38;D Systems</td>
			<td>#AF1759</td>
			<td>Primary antibody for IF</td>
		</tr>
		<tr>
			<td>p63&#945; antibody</td>
			<td>Cell Signaling Technology</td>
			<td>#ACI3066A</td>
			<td>Primary antibody for IF</td>
		</tr>
		<tr>
			<td>p63-&#945; (D2K8X) XP Rabbit mAb (PE Conjugate)</td>
			<td>Cell Signaling Technology</td>
			<td>#56687</td>
			<td>p63-&#945; PE-conjugated antibody for flow cytometry</td>
		</tr>
		<tr>
			<td>PAX6 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>#HPA030775</td>
			<td>Primary antibody for IF</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Lonza</td>
			<td>#17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>#158127</td>
			<td>Cell fixative for IF</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>#P36931</td>
			<td>DAPI mountant for hard mounting for IF</td>
		</tr>
		<tr>
			<td>PSC Cryopreservation Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>#A2644601</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Select Enzyme</td>
			<td>Gibco</td>
			<td>#12563-011</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut Dulbecco&#8217;s modified Eagle&#8217;s medium</td>
			<td>Gibco</td>
			<td>#10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut SR XenoFree CTS</td>
			<td>Gibco</td>
			<td>#10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids</td>
			<td>Gibco</td>
			<td>#11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>SB-505124</td>
			<td>Sigma-Aldrich</td>
			<td>#S4696</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>#T8787</td>
			<td>Permeabilization agent for IF</td>
		</tr>
		<tr>
			<td>VectaShield</td>
			<td>Vector Laboratories</td>
			<td>#H-1200</td>
			<td>DAPI mountant for liquid mounting for IF</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytocentrifuge, e.g. CellSpin II</td>
			<td>Tharmac</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer, e.g. BD Accuri C6</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope, e.g.Olympus IX 51</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient and scalable directed differentiation of clinically compatible corneal limbal epithelial stem cells from human pluripotent stem cells

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).

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...

Watch this Scientific Journal Video about Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells at JoVE.com

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

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.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

This protocol introduced a defined feeder cell free method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells. Limbal epithelial stem cells are responsible for renewing the corneal epithelial in the healthy eye. Damage to these cells leads to a condition known as limbal stem cell deficiency, and to the loss of corneal clarity.The cell culture methods shown here enable efficient production of limbal epithelial stem cells for future corneal cell replacement therapies. To begin, passage and maintain the feeder free human pluripotent stem cell culture as outlined in the text protocol. Make sure to use only high quality human pluripotent stem cells as starting material for differentiations.When ready to induce embryoid body formation, warm all of the needed materials and reagents to room temperature in a laminar flow hood. Detach the feeder free human pluripotent stem cells to the suspension by adding 500 microliters of xeno free trypsin EDTA to each well. An incubating at 37 degrees Celsius, with 5%CO2.After three minutes, remove the cells from the incubator and check the cell morphology to determine the optimal phase for trypsin removal. Carefully observe the cells to determine the optimal time to remove the xeno free tryspin EDTA, when the cells have rounded up, but have not detached completely. At the optimal time, remove the xeno free trypsin EDTA from the cells.Add defined trypsin inhibitor and gently pipette to detach the cells. Collect the single cell suspension in a 50 milliliter centrifuge tube. Centrifuge at 300g for five minutes.Then, remove the supernatant, and resuspend the cells in one milliliter of culture medium. After counting the cells, distribute two to three million cells in three milliliters of basal induction medium supplemented with five micromolar blebbistatin to each well of a low attachment six well plate. Incubate overnight at 37 degrees Celsius with 5%CO2.The next day, check the quality of the embryoid bodies under a microscope. Remove the medium, and replace it with three milliliters of basal induction medium supplemented with 10 micromolar SB-505124 and 50 nanograms per milliliter of human basic fiberglass growth factor. Continue incubating at 37 degrees Celsius with 5%CO2.The following two days, remove the medium, and replace it with three milliliters of basal induction medium, supplemented with 25 nanograms per milliliter of bone morphogenetic protein four. Then, continue incubation using the previous conditions. On day four, prepare a mixture of 0.5 micrograms per square centimeter of Laminin-521, and five micrograms per square centimeter of human placental collagen type four, diluted in DPBS containing calcium two and magnesium two ions.Using this mixture, coat the 100 millimeter tissue culture dishes for adherent differentiation in total coating volume of five milliliters per dish. Seal the plates, and store them at four degrees Celsius overnight. On day five, warm all of the needed materials and reagents to room temperature in a laminar flow hood.After this, remove the coating solution and add 10 milliliters of pre-warmed differentiation medium to each dish. Using a pipette, transfer the embryoid bodies from a single plate well across two to three tissue culture dishes. Then, gently shake each culture dish to evenly distribute the embryoid bodies.Maintain the cells in adherent culture at 37 degrees Celsius with 5%CO2 for the next two and a half to three weeks, making sure to replace the medium with 10 milliliters of fresh differentiation medium three times per week. Use a phase contrast microscope to regularly check the cells for the emergence of correct epithelial morphology. To begin, pre-warm all of the needed materials and reagents to room temperature in a laminar flow hood, except for the cryopreservation medium, which should be pre-chilled.Next, detach the human pluripotent stem cell derived limbal epithelial stem cells to single cell suspension by adding xeno free trypsin EDTA and incubating at 37 degrees Celsius with 5%CO2. After five minutes, remove the cells from the incubator and check the cell morphology to determine the optimal phase for trypsin removal. Carefully observe the cells to determine the optimal time to remove the xeno free trypsin EDTA, when the cells have rounded up, but have not detached completely.At the optimal time, remove the xeno free trypsin EDTA from the cells, and detach the cells as described earlier. After counting the cells, centrifuge them at 300g for five minutes. Aspirate the medium, and resuspend the cells in pre-chilled xeno free cryopreservation medium.Using a pipette, transfer the single cell suspension into cryo-tubes so that each cryo-tube contains between 500, 000 to one million cells in one milliliter of cryopreservation medium. Place the tubes in a freezing container. Within five minutes, transfer them to a freezer at negative 80 degrees Celsius for overnight storage.The next day, transfer the tubes to liquid nitrogen for long term storage. Before thawing the cells, coat all needed dishes and plate wells with a mixture of five micrograms per square centimeter human placental collagen type four, and 0.5 micrograms per square centimeter of LM-521, and pre-warm all of the needed materials and reagents to room temperature in the laminar flow hood. Next, remove the coating solution from the dishes and plate wells and add the appropriate volume of pre-warmed differentiation medium.Add pre-warmed differentiation medium to a 15 milliliter conical tube. Thaw the cells quickly to room temperature. Once thawed, immediately transfer the cell suspension to the conical centrifuge tube.Centrifuge at 300g for five minutes. Aspirate the medium, and resuspend the cell pellet in differentiation medium to remove any cryopreservation medium. Plate the cells onto precoated dishes in differentiation medium at a density of 40, 000 to 50, 000 cells per square centimeter.Maintain the cells at 37 degrees Celsius at 5%CO2, replacing the differentiation medium three times a week. On Laminin-521, the undifferentiated high quality feeder free human pluripotent stem cells first form distinct colonies with sharp edges, which merge to homogenous monolayers upon confluence. Culturing in basal induction medium, supplemented with five micromolar blebbistatin for 24 hours typically produces a suspension of tight, regular embryoid bodies of various sizes, while the embryoid body morphology should not change dramatically during the surface ectodermal induction in suspension, colonial outgrowth is seen to appear soon after the embryoid bodies are plated onto the collagen type four and Laminin-521 combination matrix in differentiation medium.Within 21 to 25 days of differentiation, the cells form confluent homogenous layers, with polygonal morphology, that is typical to epithelial cells. The cells may be then cryo stored for later use, as viability and morphology are well preserved after thawing. At day 24 of differentiation, the vast majority of the cells expressed paired box protein, PAX6, the key regulator of eye development, as well as p63 alpha, the widely recognized LESC marker.Delta Np63 is coexpressed in most of the p63 alpha positive cells, confirming the most cornea specific delta Np63 alpha positive cell phenotype. Other markers are expressed in part, while others are undetectable at this point, indicating differentiation has progressed toward the unipotent limbal epithelial progenitors, but the terminal differentiation into mature corneal epithelial cells has not yet occurred. On average, each undifferentiated feeder free human pluripotent stem cell generates 0.7 cells by day 25.At least 65%delta Np63 alpha positive cell population can be expected by day 24. Cryopreservation further purifies the cell population. Overall, the methods presented here are relatively simple, but there are a few critical points to success.High quality of the starting material is essential, as well as gentle, fluent cell culture techniques in general. This protocol provides robust means to produce limbal epithelial stem cells for clinical applications, as well as for various research purposes. Moreover, the method can be easily modified for differentiation of retinal pigment epithelial cells.

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JoVE Journal

Developmental Biology

7.5K visualizações.  University of Tampere. Este protocolo introduziu um método livre de células alimentadores definidos para diferenciar células-tronco epiteliais membros da córnea de células-tronco pluripotentes humanas. As células-tronco epiteliais limbais são responsáveis pela renovação da epitelial córnea no olho saudável. Danos a essas células levam a uma condição conhecida como deficiência de células-tronco limbais, e à perda de clareza corneal.Os métodos de cultura celular aqui mostrados permitem a pro...