Directing stem cells into the keratinocyte lineage in vitro (original) (raw)
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Cultivation of human keratinocyte stem cells: current and future clinical applications
Medical & Biological Engineering & Computing, 1998
Cultured human keratinocytes have a wide spectrum of clinical applications. Clinical results reported by several investigators are, however, contradictory. In this review, the authors discuss the biological and surgical issues which play a key role in the clinical outcome of cultured epidermal autografts used for the treatment of massive full-thickness burns. The importance of cultivation of epidermal stern cells and of their transplantation onto a wound bed prepared with donor dermis is emphasised. The paper also reviews recent data showing that: (i) cultured epidermal autografts bearing melanocytes can be used for the treatment of stable vitiligo; (ii) keratinocytes isolated from other lining epithelia, such as oral, urethral and corneal epithelia, can be cultivated and grafted onto patients suffering from disabling epithelial defects; (iii) keratinocyte stem cells can be stably transduced with retroviral vectors and are therefore attractive targets for the gene therapy of genodermatoses.
Reconstituted Skin from Murine Embryonic Stem Cells
Current Biology, 2003
cytokeratin-14 (K14) intermediate filament. Relative quantification was done by careful observation of the 1 INSERM U385 06107 Nice fields under a fluorescent microscope (Table 1). Rare induction was observed when ES cells were cultured on 2 INSERM/UMRS 514 51100 Reims gelatin. A higher but still low number of K14-positive colonies were observed in the presence of matrix de-3 CNRS UMR 6543 06108 Nice rived from SCC25 (epithelial tumor cell line derived from human tongue), 804G (epithelial cell line derived from France a rat bladder carcinoma), and MCF-10A (immortalized murine mammary epithelial) cells (Table 1). However, significant keratinocyte differentiation was obtained Summary when ES cells were seeded on human normal fibroblasts (HNF) (Figures 1Aa and 1Ab) or mouse NIH-3T3 fibro-Embryonic stem (ES) cell lines can be expanded indefinitely in culture while maintaining their potential to blast cell-secreted ECM (Table 1). The molecular basis of keratinocyte differentiation induction by the feeder differentiate into any cell type [1, 2]. During embryonic development, the skin forms as a result of reciprocal matrix remains to be understood, but it must be noted that the efficient matrices were derived from cells of interactions between mesoderm and ectoderm [3].
Experimental Dermatology, 2005
Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated; and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.
Simultaneous Isolation of Three Different Stem Cell Populations from Murine Skin
PLOS ONE, 2015
The skin is a rich source of readily accessible stem cells. The level of plasticity afforded by these cells is becoming increasingly important as the potential of stem cells in Cell Therapy and Regenerative Medicine continues to be explored. Several protocols described single type stem cell isolation from skin; however, none of them afforded simultaneous isolation of more than one population. Herein, we describe the simultaneous isolation and characterization of three stem cell populations from the dermis and epidermis of murine skin, namely Epidermal Stem Cells (EpiSCs), Skin-derived Precursors (SKPs) and Mesenchymal Stem Cells (MSCs). The simultaneous isolation was possible through a simple protocol based on culture selection techniques. These cell populations are shown to be capable of generating chondrocytes, adipocytes, osteocytes, terminally differentiated keratinocytes, neurons and glia, rendering this protocol suitable for the isolation of cells for tissue replenishment and cell based therapies. The advantages of this procedure are far-reaching since the skin is not only the largest organ in the body, but also provides an easily accessible source of stem cells for autologous graft.
Skin Stem Cells; Definition, Function, Importance and Methods of Isolation
Stem cells (SCs) are a population of undifferentiated cells with high self-renewing and differentiation potency. On the basis of origin, SCs are divided into four main groups: embryonic stem cells (ESCs), fetal stem cells (FSCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs). Interestingly, in different literatures, ASCs are considered as unipotent progenitor cells, multipotent stem cells or even pluripotent stem cells with variety of differentiation potential. ASCs reside in many adult tissues such as liver, bone marrow, adipose tissue, neural tissues, skin and etc. Among adult tissues, skin is considered as a fast self-renewing tissue which is capable to reconstruct itself during skin homeostasis and injuries. In fact, skin is mentioned as a pool of different types of SCs including keratinocyte stem cells (KSCs), hair follicle stem cells (HFSCs) and sebaceous gland stem cells (SGSCs). During skin regeneration, cooperation between these stem cells is essential for reconstruction of skin. Among these SCs, KSCs are most common cells in epidermis layer (mostly in basal layer) which are the important population of SCs for regeneration of epidermis. Herein, we reviewed different methods for skin stem cells isolation and characterization, and their potential for clinical application
Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes
Nature Biotechnology, 2008
We cultured keratinocytes from a normal human foreskin (age 4 years) in serum-free and low-calcium medium, which promotes a highly proliferative, undifferentiated state. Under these conditions, only keratin-positive keratinocytes grow (see below), and, upon addition of physiological levels of calcium, they readily differentiate and can form a fully differentiated stratified epidermis within a week (data not shown, see also ref. 9). N-terminal FLAG-tagged versions of human OCT4, SOX2, KLF4, c-MYC as well as GFP were cloned into a murine stem cell virus (MSCV)-derived retroviral vector, which provides higher transcriptional activation than the commonly used Moloney murine leukemia virus-based vectors 10 . We optimized the retroviral transduction of keratinocytes with GFP and found that two 45-min spinfections (centrifugation of cell culture plates in the presence of virus) 24 h apart resulted in nearly 100% infection of undifferentiated cells and . We then used this infection protocol to transduce keratinocytes with retroviruses encoding the four factors. Fifty-thousand passage-1 to passage-5 keratinocytes were seeded (day 0) and infected on day 1 and 2 with a 1:1:1:1 mixture of retroviruses. Cells were trypsinized on day 4 and seeded onto a layer of irradiated mouse embryonic fibroblasts (MEFs) in embryonic stem (ES) cell medium.
Immortalized keratinocyte lines derived from human embryonic stem cells
Proceedings of the National Academy of Sciences, 2006
Cells of the human embryonic stem (hES) cell line H9, when cultured in the form of embryoid bodies, give rise to cells with markers of the keratinocyte of stratified squamous epithelia. Keratinocytes also form in nodules produced in scid mice by injected H9 cells; the hES-derived keratinocytes could be recovered in culture, where their colonies underwent a peculiar form of fragmentation. Whether formed from embryoid bodies or in nodules, hES-derived keratinocytes differed from postnatal keratinocytes in their much lower proliferative potential in culture; isolated single keratinocytes could not be expanded into mass cultures. Although their growth was not improved by transduction with the hTERT gene, these keratinocytes were immortalized by transduction with the E6E7 genes of HPV16. Clonally derived lines isolated from E6E7-transduced keratinocytes continued to express markers of the keratinocyte lineage, but the frequency with which they terminally differentiated was reduced compared with keratinocytes cultured from postnatal human epidermis. If other hESderived somatic cell types also prove to be restricted in growth potential, not identical to the corresponding postnatal cell types, and to require immortalization for clonal isolation and expansion, these properties will have to be considered in planning their therapeutic use.