Generation of transgene-free hematopoietic stem cells from human induced pluripotent stem cells.

Hematopoietic stem cells (HSCs) are the rare cells responsible for the lifelong curative effects of hematopoietic cell (HC) transplantation. The demand for clinical-grade HSCs has increased significantly in recent decades, leading to major difficulties in treating patients. A promising but not yet achieved goal is the generation of HSCs from pluripotent stem cells. Here, we have obtained vector- and stroma-free transplantable HSCs by differentiating human induced pluripotent stem cells (hiPSCs) using an original one-step culture system. After injection into immunocompromised mice, cells derived from hiPSCs settle in the bone marrow and form a robust multilineage hematopoietic population that can be serially transplanted. Single-cell RNA sequencing shows that this repopulating activity is due to a hematopoietic population that is transcriptionally similar to human embryonic aorta-derived HSCs. Overall, our results demonstrate the generation of HSCs from hiPSCs and will help identify key regulators of HSC production during human ontogeny.

Different strains of Propionibacterium acnes modulate differently the cutaneous innate immunity.

Acne is a chronic inflammatory illness of the pilosebaceous follicle where innate immunity plays a central role. In acne, the density of Propionibacterium acnes is increased in the pilosebaceous unit. We hypothesized that the severity of acne is not only dependent on the proliferation of P. acnes but also dependent on the pro-inflammatory potential of P. acnes strains and consequently constitutes potential triggering factor for acne scarring. We investigated pro-inflammatory potential of five different strains of P. acnes and P. avidum in skin explants and the preventive effect of zinc gluconate. The expression of immune markers was studied by immunohistochemistry, RT-qPCR and ELISA. P. acnes strains modulate differently the expression of immune markers both at gene and at protein levels. P. acnes type III had the highest pro-inflammatory potential by up-regulating the expression of PAR-2, TNF-alpha, MMP-13 and TIMP-2, whereas P. avidum had the weakest by up-regulating only MMP-13 and TIMP-2. Preincubation of zinc gluconate, which is a modulator of innate immunity, down-regulates the expression of most immune markers induced by P. acnes, PAR-2, TIMP-2, up-regulates MMP-1, TIMP-1. Our results demonstrate that different P. acnes strains have different inflammatory potential targeting markers of cutaneous innate immunity, and that inflammatory potential can be down-regulated by zinc gluconate. As such, the inflammatory potential of P. acnes strains on acne skin may influence the severity of inflammatory acne lesions and scars.

Fetal fibroblasts and keratinocytes with immunosuppressive properties for allogeneic cell-based wound therapy.

Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.

The PI3K/AKT signaling pathway controls the quiescence of the low-Rhodamine123-retention cell compartment enriched for melanoma stem cell activity.

Melanoma is one of the most aggressive and extremely resistant to conventional therapies neoplasms. Recently, cellular resistance was linked to the cancer stem cell phenotype, still controversial and not well-defined. In this study, we used a Rhodamine 123 (Rh123) exclusion assay to functionally identify stem-like cells in metastatic human melanomas and melanoma cell lines. We demonstrate that a small subset of Rh123-low-retention (Rh123(low)) cells is enriched for stem cell-like activities, including the ability to self-renew and produce nonstem Rh123(high) progeny and to form melanospheres, recapitulating the phenotypic profile of the parental tumor. Rh123(low) cells are relatively quiescent and chemoresistant. At the molecular level, we show that melanoma Rh123(low) cells overexpress HIF1α, pluripotency factor OCT4, and the ABCB5 marker of melanoma stem cells and downregulate the expression of Cyclin D1 and CDK4. Interestingly, a short treatment with LY294002, an inhibitor of the PI3K/AKT pathway, specifically reverts a subset of Rh123(high) cells to the Rh123(low) phenotype, whereas treatment with inhibitors of mammalian target of rapamycin, phosphatase and tensin homolog or mitogen-activated protein kinase signaling does not. This phenotypic switching was associated with reduced levels of the HIF1α transcript and an increase in the level of phosphorylated nuclear FOXO3a preferentially in Rh123(low) cells. Moreover, the Rh123(low) cells became less quiescent and displayed a significant increase in their melanosphere-forming ability. All the above indicates that the Rh123(low) melanoma stem cell pool is composed of cycling and quiescent cells and that the PI3K/AKT signaling while maintaining the quiescence of Rh123(low) G0 cells promotes the exit of cycling cells from the stem cell compartment.

Human ß-defensin-2 and psoriasin, two new innate immunity targets of zinc gluconate.

BACKGROUND: Antimicrobial peptides (AMPs) are a large family of peptides implicated in innate immunity, especially in the epidermis. Zinc gluconate has been proven to be efficient to treat inflammatory dermatoses, such as acne vulgaris. OBJECTIVES: The aim of our work was to determine whether AMPs could be new targets of zinc gluconate treatment in inflammatory dermatoses. MATERIAL AND METHODS: To test this hypothesis, we used an ex vivo lipopolysaccharide (LPS)-induced inflammatory skin explant model, with or without zinc gluconate pretreatment. We evaluated human ß-defensin-2 (hBD-2), human ß-defensin-4 (hBD-4) and psoriasin protein expression and release by immunohistochemistry and ELISA, as well as the mRNA expression level by quantitative PCR. RESULTS: We found that hBD-2 and psoriasin mRNA expression levels and hBD-2 extracellular release, but not hBD-4 expression and release, were significantly upregulated by zinc gluconate in LPS-stimulated inflammatory skin explants. CONCLUSION: These results suggest that hBD-2 and psoriasin may be two main targets of zinc gluconate, involved in its anti-inflammatory activity in dermatoses.

Ex vivo demonstration of a synergistic effect of Adapalene and benzoyl peroxide on inflammatory acne lesions.

Acne is a chronic inflammatory disease of the pilosebaceous follicle. Thanks to its ability to reduce both comedones and inflammatory lesions, the association of a retinoid and benzoyl peroxide (BPO) is now recommended for the treatment of acne. However, the mechanisms of action of this combined therapy on inflammatory acne lesions are not well understood. In an ex vivo immunohistochemistry study, we investigated the potential synergistic modulator effect of Adapalene associated with BPO on keratinocytes proliferation/differentiation and innate immunity in inflammatory acne lesions. We demonstrated that proliferation (Ki-67), adhesion/differentiation (integrin α(2), α(3) and α(6)) and innate immunity (TLR-2, ß-defensin 4, IL-8) markers are overexpressed in inflammatory acne skin compared with uninvolved acne skin. Association of Adapalene and BPO significantly decreased expression of Ki67, α(2) and α(6) integrins, TLR-2, ß-defensin 4 and IL-8 in inflammatory acne skin, whereas single treatments with Adapalene or BPO alone were less effective. These results contribute to explain the comedolytic and anti-inflammatory activities of this combined therapy observed in recent clinical trials.

Value of large scale expansion of tumor infiltrating lymphocytes in a compartmentalised gas-permeable bag: interests for adoptive immunotherapy.

BACKGROUND: Adoptive cell therapy (ACT) has emerged as an effective treatment for patients with metastatic melanoma. However, there are several logistical and safety concerns associated with large-scale ex vivo expansion of tumour-specific T lymphocytes for widespread availability of ACT for cancer patients. To address these problems we developed a specific compartmentalised bag allowing efficient expansion of tumour-specific T lymphocytes in an easy handling, closed system. METHODS: Starting from lymph nodes from eight melanoma patients, we performed a side-by-side comparison of Tumour-Infiltrating Lymphocytes (TIL) produced after expansion in the compartmentalised bag versus TIL produced using the standard process in plates. Proliferation yield, viability, phenotype and IFNγ secretion were comparatively studied. RESULTS: We found no differences in proliferation yield and cell viability between both TIL production systems. Moreover, each of the cell products complied with our defined release criteria before being administered to the patient. The phenotype analysis indicated that the compartmentalised bag favours the expansion of CD8+ cells. Finally, we found that TIL stimulated in bags were enriched in reactive CD8+ T cells when co-cultured with the autologous melanoma cell line. CONCLUSIONS: The stimulation of TIL with feeder cells in the specifically designed compartmentalised bag can advantageously replace the conventional protocol using plates. In particular, the higher expansion rate of reactive CD8+ T cells could have a significant impact for ACT.

Asymmetric distribution of epidermal growth factor receptor directs the fate of normal and cancer keratinocytes in vitro.

Cancer cells are unequal in a tumor mass and in established cultures. This is attributable to cancer stem cells with the unique ability to self-renew and to generate differentiating progeny. This ability is controlled at the level of asymmetric division by mechanisms that are yet not well defined. We found that normal and cancer keratinocyte fate was linked to the asymmetric distribution of epidermal growth factor receptor (EGFR) during mitosis. Although essential for epithelial cell proliferation, differentiation, and survival, this receptor was not present on the surface of cells satisfying criteria for stem cells such as quiescence, competence to produce functionally distinct daughters, high proliferative and clonogenic potential, sphere formation ability, and expression of stem cell markers. In contrast, keratinocytes displaying EGFR acquired a more differentiated phenotype, suggesting that EGFR may be involved in a switch from stem to transient amplifying cell fate. This switch was associated with changes in the expression profile of cell cycle, survival, and mitochondria controlling proteins that varied between normal and cancer cells. In conclusion, it appears that an unequal distribution of EGFR at mitosis controls keratinocyte fate by balancing quiescence and cycling of EGFR(-) cells, clearly malfunctioning in cancer. We believe that our findings provide mechanistic insights into the development of resistance to anti-EGFR therapies.

Human dental pulp stem cells differentiate into neural crest-derived melanocytes and have label-retaining and sphere-forming abilities.

Adult tissues contain highly proliferative, clonogenic cells that meet criteria of multipotent stem cells and are potential sources for autologous reparative and reconstructive medicine. We demonstrated that human dental pulp contains self renewing human dental pulp stem cells (hDPSCs) capable of differentiating into mesenchymal-derived odontoblasts, osteoblasts, adipocytes, and chondrocytes and striated muscle, and interestingly, also into non-mesenchymal melanocytes. Furthermore, we showed that hDPSC cultures include cells with the label-retaining and sphere-forming abilities, traits attributed to multipotent stem cells, and provide evidence that these may be multipotent neural crest stem cells.

Variable Bax antigenicity is linked to keratinocyte position within epidermal strata and UV-induced apoptosis.

Pro- and anti-apoptotic members of the Bcl-2 family are fundamental in the control of apoptosis. Among them, Bax plays a key role in apoptosis induction by mediating the release of apoptogenic factors from mitochondria to the cytosol. In this report, we investigated, by immunohistofluorescence, the in vivo distribution of Bax in normal human epidermis before and 24 h after exposure to solar-simulated radiation. Bax expression was evaluated with three different, Western blot pretested, anti-Bax antibodies (Ab) and correlated with markers of keratinocyte differentiation and apoptosis using anti-beta(1) integrin and anti-active caspase-3 Abs respectively. Using anti-Bax N20 and A-3533 polyclonal Ab, we found that, whereas undifferentiated keratinocytes of the basal proliferative compartment contained Bax in the cytosol, the differentiated suprabasal cells had Bax mainly in the nucleus. This immunoreactivity pattern was not modified by skin irradiation. Interestingly, the well known apoptosis-related Bax redistribution to mitochondria in response to a cell death signal, could be detected only with yet another, the 2D2 monoclonal Ab. This relocalization occurred specifically in apoptotic, active caspase-3 positive cells of irradiated epidermis. Our data highlight the differentiation- and apoptosis-associated changes in the pattern of Bax subcellular and cellular distribution as uncovered by different anti-Bax Abs and suggest that Bax undergoes successive activation that progresses in parallel with keratinocyte differentiation and apoptosis.

Hydrogen peroxide-induced cell death in normal human keratinocytes is differentiation dependent.

More than other tissues, skin is exposed to numerous external stresses generating ROS that, in addition to endogenous oxygen radicals, cause keratinocyte alterations and contribute in part to photocarcinogenesis and aging. Recent evidence suggests a differentiation-dependent susceptibility of keratinocytes to apoptosis. We explored hydrogen peroxide-induced cell death in normal human keratinocytes according to their differentiation. On H(2)O(2)-exposed skin explants, caspase-3 was strongly activated in basal keratinocytes double stained with beta(1) integrin, whereas DNA fragmentation occurred in suprabasal cells only without caspase-3 activation. In addition, isolated basal keratinocytes, selected by adhesion to type IV collagen, were more sensitive than nonadherent cells to H(2)O(2)-induced apoptosis with regard to mitochondrial transmembrane potential (Deltapsi(mt)) collapse and membrane integrity. Similarly, necrotic/late apoptotic cells were present at low levels only in the adherent epidermal population. Furthermore, in primary cultures of undifferentiated keratinocytes H(2)O(2)-induced cell death appeared via a mitochondrial failure. Deltapsi(mt) collapse was associated with a strong early activation of the initiatory caspase-8, then the executive caspase-3, and, to a lesser extent, the inflammatory caspase-1. Finally, undifferentiated basal cells possess a higher sensitivity than differentiated suprabasal cells to H(2)O(2)-induced cell death, and apoptosis in human keratinocytes occurs via different pathways depending on the cell's differentiation state.

Sensitive and reliable JC-1 and TOTO-3 double staining to assess mitochondrial transmembrane potential and plasma membrane integrity: interest for cell death investigations.

BACKGROUND: Apoptosis is currently studied by flow cytometry with mitochondrial membrane potential (Deltapsimt) and membrane integrity fluorochromes. Rhodamine 123 and DiOC6(3) remain controversial to identify cells displaying a low Deltapsimt. JC-1 constitutes a good Deltapsimt indicator, due to a fluorescence shift from green to orange emission, according to the increase in Deltapsimt. Nevertheless, it is not feasible to analyze it simultaneously with propidium iodide. Among available fluorescent probes, TOTO-3 seems to be a good candidate for double staining with JC-1. METHODS: Cell death of HaCaT cells was induced by H2O2 and FasL. Samples were stained with DiOC6(3)/IP or JC-1/TOTO-3 then analyzed by flow cytometry. Results were supported by confocal microscopy analyses of mitochondrial membrane potential. Moreover, cell morphology was determined on the sorted subpopulations defined on the basis of staining (JC-1 versus TOTO-3). RESULTS: We found that JC-1 is a more efficient mitochondrial probe than DiOC6(3). After stress induction, the fluorescence level of JC-1 and TOTO-3 clearly defined three fluorescent subpopulations, respectively: (1) JC-1high and TOTO-3low, (2) JC-1low and TOTO-3medium, and (3) JC-1low and TOTO-3high. Their morphologic aspects after cell sorting indicated that they corresponded to three functional states (intact, apoptotic, and necrotic cells), and data were supported by caspase activity measurements. CONCLUSIONS: We propose a reliable and efficient staining, with JC-1 and TOTO-3 to discriminate three functional cellular states: intact, apoptotic, and necrotic/late apoptotic cells by flow cytometry.