BackgroundHuman pluripotent stem cells (hPSCs) provide a promising cell source for ocular cell replacement therapy, but often lack standardized and xenogeneic-free culture and differentiation protocols. We aimed to develop a xeno- and feeder cell-free culture system for undifferentiated hPSCs along with efficient methods to derive ocular therapy target cells: retinal pigment epithelial (RPE) cells and corneal limbal epithelial stem cells (LESCs).MethodsMultiple genetically distinct hPSC lines were adapted to a defined, xeno-, and feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial cells were established utilizing xeno-free media and a combination of extracellular matrix proteins. Both differentiation methods shared the same basal elements, using only minor inductive modifications during early differentiation towards desired cell lineages. The resulting RPE cells and LESCs were characterized after several independent differentiation experiments and recovery after xeno-free cryopreservation.ResultsThe defined, xeno-, and feeder-free culture system provided a robust means to generate high-quality hPSCs with chromosomal stability limited to early passages. Inductive cues introduced during the first week of differentiation had a substantial effect on lineage specification, cell survival, and even mature RPE properties. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to cell populations expressing LESC markers such as p40/p63α by day 24. Finally, we established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE cells, and hPSC-LESCs, and demonstrated successful recovery after thawing.ConclusionsWe propose methods for efficient and scalable, directed differentiation of high-quality RPE cells and LESCs. The two clinically relevant cell types are generated with simple inductive modification of the same basal method, followed by adherent culture, passaging, and cryobanking.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0738-4) contains supplementary material, which is available to authorized users.
Background The differentiation of corneal limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) has great power as a novel treatment for ocular surface reconstruction and for modeling corneal epithelial renewal. However, the lack of profound understanding of the true LSC population identity and the regulation of LSC homeostasis is hindering the full therapeutic potential of hPSC-derived LSCs as well as primary LSCs. Methods The differentiation trajectory of two distinct hPSC lines towards LSCs was characterized extensively using immunofluorescence labeling against pluripotency, putative LSC, and mature corneal epithelium markers. Cell counting, flow cytometry, and qRT-PCR were used to quantify the differences between distinct populations observed at day 11 and day 24 time points. Initial differentiation conditions were thereafter modified to support the maintenance and expansion of the earlier population expressing ABCG2. Immunofluorescence, qRT-PCR, population doubling analyses, and transplantation into an ex vivo porcine cornea model were used to analyze the phenotype and functionality of the cell populations cultured in different conditions. Results The detailed characterization of the hPSC differentiation towards LSCs revealed only transient expression of a cell population marked by the universal stemness marker and proposed LSC marker ABCG2. Within the ABCG2-positive population, we further identified two distinct subpopulations of quiescent ∆Np63α-negative and proliferative ∆Np63α-positive cells, the latter of which also expressed the acknowledged intestinal stem cell marker and suggested LSC marker LGR5. These populations that appeared early during the differentiation process had stem cell phenotypes distinct from the later arising ABCG2-negative, ∆Np63α-positive third cell population. Importantly, novel culture conditions modulating the Wnt and BMP signaling pathways allowed efficient maintenance and expansion of the ABCG2-positive populations. In comparison to ∆Np63α-positive hPSC-LSCs cultured in the initial culture conditions, ABCG2-positive hPSC-LSCs in the novel maintenance condition contained quiescent stem cells marked by p27, demonstrated notably higher population doubling capabilities and clonal growth in an in vitro colony-forming assay, and increased regenerative potential in the ex vivo transplantation model. Conclusions The distinct cell populations identified during the hPSC-LSC differentiation and ABCG2-positive LSC maintenance may represent functionally different limbal stem/progenitor cells with implications for regenerative efficacy. Electronic supplementary material The online version of this article (10.1186/s13287-019-1354-2) contains supplementary material, which is available to authorized users.
Background Differentiation of functional limbal stem cells (LSCs) from human pluripotent stem cells (hPSCs) is an important objective which can provide novel treatment solutions for patients suffering from limbal stem cell deficiency (LSCD). Yet, further characterization is needed to better evaluate their immunogenicity and regenerative potential before clinical applications. Methods Human PSCs were differentiated towards corneal fate and cryopreserved using a clinically applicable protocol. Resulting hPSC-LSC populations were examined at days 10–11 and 24–25 during differentiation as well as at passage 1 post-thaw. Expression of cornea-associated markers including PAX6, ABCG2, ∆Np63α, CK15, CK14, CK12 and ABCB5 as well as human leukocyte antigens (HLAs) was analyzed using immunofluorescence and flow cytometry. Wound healing properties of the post-thaw hPSC-LSCs were assessed via calcium imaging and scratch assay. Human and porcine tissue-derived cultured LSCs were used as controls for marker expression analysis and scratch assays at passage 1. Results The day 24–25 and post-thaw hPSC-LSCs displayed a similar marker profile with the tissue-derived LSCs, showing abundant expression of PAX6, ∆Np63α, CK15, CK14 and ABCB5 and low expression of ABCG2. In contrast, day 10–11 hPSC-LSCs had lower expression of ABCB5 and ∆Np63α, but high expression of ABCG2. A small portion of the day 10–11 cells coexpressed ABCG2 and ABCB5. The expression of class I HLAs increased during hPSC-LSCs differentiation and was uniform in post-thaw hPSC-LSCs, however the intensity was lower in comparison to tissue-derived LSCs. The calcium imaging revealed that the post-thaw hPSC-LSCs generated a robust response towards epithelial wound healing signaling mediator ATP. Further, scratch assay revealed that post-thaw hPSC-LSCs had higher wound healing capacity in comparison to tissue-derived LSCs. Conclusions Clinically relevant LSC-like cells can be efficiently differentiated from hPSCs. The post-thaw hPSC-LSCs possess functional potency in calcium responses towards injury associated signals and in wound closure. The developmental trajectory observed during hPSC-LSC differentiation, giving rise to ABCG2+ population and further to ABCB5+ and ∆Np63α+ cells with limbal characteristics, indicates hPSC-derived cells can be utilized as a valuable cell source for the treatment of patients afflicted corneal blindness due to LSCD. Graphical Abstract
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.