Dataset Information

Differentiation of adipose-derived stem cells to functional CD105^neg CD73^low melanocyte precursors guided by defined culture condition.

ABSTRACT:

Background

The generation of functional human epidermal melanocytes (HEM) from stem cells provides an unprecedented source for cell-based therapy in vitiligo. Despite the important efforts exerted to obtain melanin-producing cells from stem cells, pre-clinical results still lack the safety and scalability characteristics essential for their translational application.

Methods

Here, we report a rapid and efficient protocol based on defined culture conditions capable of differentiating adult adipose-derived stem cells (ADSC) to scalable amounts of proliferative melanocyte precursors (PreMel) within 30 days. PreMel were characterized in vitro through qPCR, Western blot, flow cytometry, biochemical assays, and in vivo assays in immunocompromised mice (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ, or NSG).

Results

After 30 days of differentiation, the stem cell-derived PreMel were defined as CD105^neg CD73^low according to immunophenotypic changes in comparison with parental stem cell markers. In addition, expression of microphthalmia-associated transcription factor (MITF), active tyrosinase (TYR), and the terminal differentiation-involved premelanosome protein (PMEL) were detected. Furthermore, PreMel had the potential to synthesize melanin and package it into melanosomes both in vitro and in vivo in NSG mice skin.

Conclusions

This study proposes a rapid and scalable protocol for the generation of proliferative melanocyte precursors (PreMel) from ADSC. These PreMel display the essential functional characteristics of bona fide HEM, opening a new path for an autologous cellular therapy for vitiligo patients.

SUBMITTER: Zavala G

PROVIDER: S-EPMC6688240 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Publications

Differentiation of adipose-derived stem cells to functional CD105<sup>neg</sup> CD73<sup>low</sup> melanocyte precursors guided by defined culture condition.

Zavala Gabriela G Sandoval Carolina C Meza Daniel D Contreras Rafael R Gubelin Walter W Khoury Maroun M

Stem cell research & therapy 20190809 1

<h4>Background</h4>The generation of functional human epidermal melanocytes (HEM) from stem cells provides an unprecedented source for cell-based therapy in vitiligo. Despite the important efforts exerted to obtain melanin-producing cells from stem cells, pre-clinical results still lack the safety and scalability characteristics essential for their translational application.<h4>Methods</h4>Here, we report a rapid and efficient protocol based on defined culture conditions capable of differentiati ...[more]

PMID: 31399041

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Project description:BackgroundReducing cutaneous scar formation is important for assessing the success of skin wound healing. Although it is generally accepted that adipose-derived mesenchymal stem cells (AMSCs) have substantial therapeutic potential, efforts are continuously made to improve the outcome of AMSC therapy. Post-transcriptional suppression of procollagen-lysine 1, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) in AMSCs has been shown to greatly reduce scar formation during skin wound healing, likely through modulating macrophage polarization. In the present study, we tested whether a CD73+ subpopulation of AMSCs could reduce scar formation compared with CD73- AMSCs.MethodsThe gene profile of CD73+ versus CD73- AMSCs was obtained from a validated public database, GSE167219. AMSCs were isolated from adipose tissue surrounding the groin of mice, after which CD73+ versus CD73- AMSCs were sorted using flow cytometry. PLOD1 levels were determined in CD73+ versus CD73- AMSCs. Then, PLOD1 in CD73- AMSCs was depleted by a short-hair interfering RNA against PLOD1 (sh-PLOD1), while PLOD1 in CD73+ AMSCs was increased by expression of a PLOD1 transgene. A blade was used to induce a skin injury on the middle back of the mice. Either CD73+ AMSCs or CD73+ PLOD1 AMSCs or CD73- AMSCs or CD73- sh-PLOD1 AMSCs were intravenously transplanted into the injured region of the mice. Fibrosis and the underlying mechanisms were investigated. Co-immunoprecipitation was performed to evaluate interaction between CD73 and PLOD1.ResultsCD73+ AMSCs expressed significantly lower levels of PLOD1, a potent stimulator of fibrosis, compared with CD73- AMSCs. Transplantation of CD73+ AMSCs generated significantly reduced fibrosis at the skin injury site compared with CD73- AMSCs. However, expression of PLOD1 in CD73+ AMSCs abolished its advantageous effects on fibrosis reduction, while depletion of PLOD1 in CD73- AMSCs improved the outcome of fibrosis to the levels of transplantation of CD73+ AMSCs. Co-immunoprecipitation showed no direct protein interaction between CD73 and PLOD1.ConclusionsCD73+ AMSCs are a subgroup of AMSCs with better therapeutic effects on wound healing, and can inhibit scar formation through reduced PLOD1 in an indirect manner.

Project description:Conventional CD4pos regulatory T (Treg) cells characterized by expression of the key transcription factor forkhead box P3 (FoxP3) are crucial to control immune responses, thereby maintaining homeostasis and self-tolerance. Within the substantial population of non-conventional T cell receptor (TCR)αβpos CD4negCD8αneg double-negative (dn) T cells of dogs, a novel FoxP3pos Treg-like subset was described that, similar to conventional CD4pos Treg cells, is characterized by high expression of CD25. Noteworthy, human and murine TCRαβpos regulatory dn T cells lack FoxP3. Immunosuppressive capacity of canine dn T cells was hypothesized based on expression of inhibitory molecules (interleukin (IL)-10, cytotoxic T-lymphocyte associated protein 4, CTLA4). Here, to verify their regulatory function, the dnCD25pos (enriched for FoxP3pos Treg-like cells) and the dnCD25neg fraction, were isolated by fluorescence-activated cell sorting from peripheral blood mononuclear cells (PBMC) of Beagle dogs and analyzed in an in vitro suppression assay in comparison to conventional CD4posCD25pos Treg cells (positive control) and CD4posCD25neg T cells (negative control). Canine dnCD25pos T cells suppressed the Concanavalin A-driven proliferation of responder PBMC to a similar extent as conventional CD4posCD25pos Treg cells. Albeit to a lesser extent than FoxP3-enriched dn and CD4posCD25pos populations, even dnCD25neg T cells reduced the proliferation of responder cells. This is remarkable, as dnCD25neg T cells have a FoxP3neg phenotype comparable to non-suppressive CD4posCD25neg T cells. Both, CD25pos and CD25neg dn T cells, can mediate suppression independent of cell-cell contact and do not require additional signals from CD4posCD25neg T cells to secrete inhibitory factors in contrast to CD4posCD25pos T cells. Neutralization of IL-10 completely abrogated the suppression by dnCD25pos and CD4posCD25pos Treg cells in a Transwell™ system, while it only partially reduced suppression by dnCD25neg T cells. Taken together, unique canine non-conventional dnCD25pos FoxP3pos Treg-like cells are potent suppressor cells in vitro. Moreover, inhibition of proliferation of responder T cells by the dnCD25neg fraction indicates suppressive function of a subset of dn T cells even in the absence of FoxP3. The identification of unique immunoregulatory non-conventional dn T cell subpopulations of the dog in vitro is of high relevance, given the immunotherapeutic potential of manipulating regulatory T cell responses in vivo.

Dataset Information

Differentiation of adipose-derived stem cells to functional CD105^neg CD73^low melanocyte precursors guided by defined culture condition.

Background

Methods

Results

Conclusions

Publications

Differentiation of adipose-derived stem cells to functional CD105<sup>neg</sup> CD73<sup>low</sup> melanocyte precursors guided by defined culture condition.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Differentiation of adipose-derived stem cells to functional CD105neg CD73low melanocyte precursors guided by defined culture condition.

Background

Methods

Results

Conclusions

Publications

Differentiation of adipose-derived stem cells to functional CD105<sup>neg</sup> CD73<sup>low</sup> melanocyte precursors guided by defined culture condition.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Differentiation of adipose-derived stem cells to functional CD105^neg CD73^low melanocyte precursors guided by defined culture condition.