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Forthcoming article in Stem Cells - From Hair to Cornea: Towards the Therapeutic Use of Hair Follicle-Derived Stem Cells in the Treatment of Limbal Stem Cell Deficiency

By Stuart P. Atkinson

Many laboratories throughout the world are working with embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) as sources of cells for replacement therapy in human disease. The necessity for patient specificity of these cells is generally a significant problem, which may be overcome in the case of iPSCs if they ultimately prove to be a suitable replacement for ESCs. However, even if they do prove to be similar, differentiation protocols are highly inefficient and require long culture periods in vitro and expensive culture conditions. Orthologous adult stem cell therapy, that is treatment of the patient with his own stem cells, is a strategy that has interested many groups and their potential for transdifferentiation and use in therapy is tantalizing.

One human disease/affliction in which orthologous adult stem cell therapy has been applied to is limbal stem cell deficiency (LSCD), which can lead to the loss of sight in the affected eye(s) (Ahmad et al). A proven therapy for unilateral disease is in vitro expansion of cells from a limbal biopsy from the non-affected eye (Kolli et al), and subsequent transplantation of the cells to the affected eye (Kolli et al). Bilateral disease entails the use of donor tissue and therefore systemic immunosuppressive therapy and the success rate of this method is significantly reduced. Research has therefore been geared towards the search for alternative autologous adult stem cell types which could potentially be used to replace bona fide limbal stem cells. Other epithelial cell types have been considered as potential substitutes, including oral mucosa epithelium, however no satisfactory long term results have so far been demonstrated.

The hair follicle (HF) has been shown to harbor mesenchymal cells which possess the ability to differentiate towards several cell lineages in vivo and in vitro and the follicle also contains a population of stem cells of epithelial origin (HFSCs). Previous studies (Blazejewska et al) have shown that HFSCs could be differentiated in vitro into cells with a corneal epithelial phenotype and now this work has moved forward to in vivo studies (Meyer-Blazejewska et al) and the results of this work are now presented in Stem Cells.

The in vivo potential of HFSCs was studied using an inducible, tissue specific triple transgenic mouse model (K12rtTA/rtTA/ROSAmTmG), in which only those cells expressing Krt12, a corneal epithelial differentiation marker, will be positive for eGFP in the presence of Dox, while in the absence of Dox, all cells express a membrane bound red fluorescent protein (mT). Vibrissae hair follicles from the transgenic mice were first dissected and the mesenchymal capsule removed to leave only the epithelial core. After a multi-step enzymatic digestion to give a single cell suspension, cells were plated on 3T3 feeders to purify and expand stem and progenitor cells by means of clonal growth. Phenotypic analysis showed that Krt15, a putative stem cell marker expressed abundantly in the murine bulge cells, was expressed in several small cell clusters within each stem cell clones, while Krt10, a marker for epidermal differentiation was scarcely expressed. After two weeks of growth, clones expressed mT, were negative for eGFP and had small, compact epithelial shaped cells, all characteristic of an undifferentiated epithelial stem cell type. At this stage the HFSC clones were transferred to fibrin carriers and cultivated until they formed a confluent cell sheet that expressed Krt15 and very small amounts of Krt10, but no Krt12 or Pax6 (another corneal epithelial differentiation marker). The carriers where then transferred (cell side first) onto the ocular surface of C57BL/6 wild type LSCD mice, to give one treated LSCD eye and one control LSCD eye.

Fluorescein uptake assays demonstrated that these HSFCs were able to resurface the cornea by four weeks after transplantation and the morphology of the treated corneal surface resembled normal corneal epithelium. Encouragingly, overall corneal reconstruction with HSFC was observed in 80% of the mice, with the other 20% containing some degree of conjunctival ingrowth. The presence of red and green cells in treated corneas showed that the transplant was maintained for at least five weeks and also that the transplant gave rise to eGFP expressing cells which were localized primarily to the basal layer of the epithelium. eGFP was first detected three days post-transplantation and reached peak expression at 14 days, with maintenance of high expression levels until the end of the experiment. The turnover rate of corneal epithelium is estimated to be 14 days, and as HFSC cells were observed from 3 days to 21-39 days this suggests that the HFSCs had successfully settled within the corneal stem cell niche and were able to give rise to cells when required. Krt10 was not expressed in the LSCD control or LSCD treated eye, while Krt4, a conjunctival marker, was highly expressed in the control eye and minimally in the treated eye and Krt15 showed wide expression in the transplanted eye, but only in the cells lacking EGFP which have not differentiated terminally, suggestive that the HFSCs can re-establish the stem cell niche.

Overall, the data suggests that when placed into the appropriate microenvironment, i.e. the ocular surface, HSFCs can differentiate along a differentiation lineage distinct from their origin, therefore providing evidence of the potential therapeutic use of these adult stem cells. However an appropriate stem cell source in humans must be identified and the authors point towards preliminary data from a pilot study suggesting that skin biopsies from the human scalp can allow for HFSC isolation and enrichment by clonal expansion. Engraftment experiments in rabbits using these cells also suggest that engraftment can be successful and further, that some cells differentiate towards a corneal epithelial phenotype.



Stem cell therapies for ocular surface disease.
Ahmad S, Kolli S, Lako M, Figueiredo F, Daniels JT.
Drug Discov Today. 2010 Apr;15(7-8):306-13

Loss of corneal epithelial stem cell properties in outgrowths from human limbal explants cultured on intact amniotic membrane.
Kolli S, Lako M, Figueiredo F, Mudhar H, Ahmad S.
Regen Med. 2008 May;3(3):329-42.

Successful clinical implementation of corneal epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency.
Kolli S, Ahmad S, Lako M, Figueiredo F.
Stem Cells. 2010 Mar 31;28(3):597-610.

Corneal limbal microenvironment can induce transdifferentiation of hair follicle stem cells into corneal epithelial-like cells.
Blazejewska, EA, Schlotzer-Schrehardt U, Zenkel M, et al.
Stem Cells 2009; 27(3): 642-652.

From Hair to Cornea: Towards the Therapeutic Use of Hair Follicle-Derived Stem Cells in the Treatment of Limbal Stem Cell Deficiency.
Meyer-Blazejewska EA, Call MK, Yamanaka O, Liu H, Schlötzer-Schrehardt U, Kruse FE, Kao WW.
Stem Cells. 2010 Oct 18