Name: efficient and scalable directed differentiation of clinically compatible corneal limbal epithelial stem cells from human pluripotent stem cells
Uploaded: 2018-10-24T14:00:00
Description: 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

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. 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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 ...

Research

JoVE Journal

Developmental Biology

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Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

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