Elsevier

Burns

Volume 32, Issue 1, February 2006, Pages 16-19
Burns

Cultured human epithelium: Human umbilical cord blood stem cells differentiate into keratinocytes under in vitro conditions

https://doi.org/10.1016/j.burns.2005.08.020Get rights and content

Abstract

Background

Stem cells have the capacity to renew or to give rise to a specialized cell types. Human umbilical cord blood (HUCB) has been explored as an alternative source of stem cells. However, its potential to differentiate into cells of other tissues is still under discussion. The aim of our study was to evaluate if HUCB stem cells could differentiate into epithelial cells under in vitro conditions.

Methods

Human keratinocytes derived from adult female skin donors, were isolated and cultured on fibrin glue/fibroblast gels—control group. In the umbilical cord blood cell group, male umbilical cord blood cells were added at a 1:10 ratio to keratinocytes and co-cultured on the fibrin glue/fibroblasts gel.

After 15 days of culture, the sheets were analyzed by use of histochemistry and FISH. DNA was extracted and evaluated by use of polymerase chain reaction (PCR) for detection of Y-chromosome-specific sequences.

Results

In both groups a regular epithelial sheet consisting of three to four layers of cells was formed. Using PCR and FISH, in the umbilical cord blood cell group the presence of Y-chromosome-specific sequences in the cultured keratinocytes could be detected. In the control group, no Y-chromosome-specific sequences could be detected.

Conclusion

Our findings indicate that umbilical cord blood stem cells differentiate into epithelial cells under in vitro conditions and thereby, might serve as a starting material for isolation and expansion of cells for transplantation in patients with large skin defects.

Introduction

Stem cells have the unique capacity to either self-renew or to give rise to a specialized cell type [1]. Bone marrow (BM) contains stem cells (haematopoietic stem cells, HSC), which differentiate into all mature blood cells and marrow stromal cells. They have the capacity to differentiate into mature cells in multiple mesenchymal tissues, including fat, bone, and cartilage [2], [3], [4]. Recent studies indicate that HSC also differentiate into cells of ectodermal, mesodermal and endodermal tissues. Human umbilical cord blood (HUCB) has been explored as an alternative source of stem cells to repopulate the BM in the treatment of diseases in children and adults [5], [6], [7]. Moreover, in contrast to BM aspiration, HUCB is obtained by a simple, safe and painless procedure. Thus, HUCB has become an indispensable source to treat hematological disorders [8], [9]. However, its potential to differentiate into cells of other tissues like skin is still under discussion.

Cultured epidermis has been used as grafting material in different clinical situations such as treatment of burn wounds [10], chronic skin ulcers [11], and oral mucous defects [12]. This method has gained attention in the treatment of seriously burned patients, since it allows grafting of an epithelial surface, large enough to cover the needs of a patient [13]. Rapid and effective burn wound closure is one of the most important aspects in the treatment of burn patients, because the patient is in a sub-septic condition, until all skin defects are closed. Unfortunately, in larger burns there is a lack of donor sites for grafting procedures.

Bearing in mind the possible use of cultured skin substitutes for the treatment of seriously burned patients, we evaluated if HUCB stem cells were capable to differentiate into epithelial cells which in turn could be transplanted onto skin defects. One major aspect was to evaluate, if they differentiated into Keratinocytes isolated under in vitro conditions. We conducted our in vitro study with female keratinocytes and male HUCB stem cells to study the possible contribution of HUCB stem cells to the regeneration of skin tissue.

Section snippets

Primary keratinocyte culture

Normal human keratinocytes derived from adult female skin donors, were isolated as previously described [14]. Briefly, a skin section was incubated in trypsin-EDTA for 18 h at 4 °C in order to obtain individual cells. Keratinocytes were then cultured on fibrin glue/fibroblasts gels as a control group. Where indicated, male umbilical cord blood cells were added at an 1:10 ratio to the keratinocytes and co-cultured on the fibrin glue/fibroblasts gel as the treatment-umbilical cord blood cell group.

Morphological and differentiation features of keratinocytes

At day 15, a regular epithelial sheet consisting of three to four layers of cells was formed. A limiting membrane demarcating the keratinocytes from the fibrin matrix was discernible. Squamous differentiation with flattening out of cells similar to Strata reticulare and corneum found in vivo were observed. Nuclei of basal cells were regularly spaced from each other and the chromatin was of homogeneous appearance without prominent nucleoli. Mitoses were rare. Between both groups no

Discussion

At the moment, several technologies are under active development to aid cutaneous wound repair [14]. However, many of them deal with a single aspect of the healing process that does not necessarily serve as a primary source of new tissue. Nowadays there has been a great deal of interest in using stem cells in tissue regeneration [16]. Stem cells are defined as cells capable to perform self-renewal and multi-lineage differentiation [17]. Embryonic stem cells are totipotent, have the potential to

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