Immunophenotyping Reveals the Diversity of Human Dental Pulp Mesenchymal Stromal Cells In vivo and Their Evolution upon In vitro Amplification

International audienceMesenchymal stromal/stem cells (MSCs) from human dental pulp (DP) can be expanded in vitro for cell-based and regenerative dentistry therapeutic purposes. However, their heterogeneity may be a hurdle to the achievement of reproducible and predictable therapeutic outcomes. To get a better knowledge about this heterogeneity, we designed a flow cytometric strategy to analyze the phenotype of DP cells in vivo and upon in vitro expansion with stem cell markers. We focused on the CD31 − cell population to exclude endothelial and leukocytic cells. Results showed that the in vivo CD31 − DP cell population contained 1.4% of CD56 + , 1.5% of CD146 + , 2.4% of CD271 + and 6.3% of MSCA-1 + cells but very few Stro-1 + cells (≤1%). CD56 + , CD146 + , CD271 + , and MSCA-1 + cell subpopulations expressed various levels of these markers. CD146 + MSCA-1 + , CD271 + MSCA-1 + , and CD146 + CD271 + cells were the most abundant DP-MSC populations. Analysis of DP-MSCs expanded in vitro with a medicinal manufacturing approach showed that CD146 was expressed by about 50% of CD56 + , CD271 + , MSCA-1 + , and Stro-1 + cells, and MSCA-1 by 15-30% of CD56 + , CD146 + , CD271 + , and Stro-1 + cells. These ratios remained stable with passages. CD271 and Stro-1 were expressed by <1% of the expanded cell populations. Interestingly, the percentage of CD56 + cells strongly increased from P1 (25%) to P4 (80%) both in all sub-populations studied. CD146 + CD56 + , MSCA-1 + CD56 + , and CD146 + MSCA-1 + cells were the most abundant DP-MSCs at the end of P4. These results established that DP-MSCs constitute a heterogeneous mixture of cells in pulp tissue in vivo and in culture, and that their phenotype is modified upon in vitro expansion. Further studies are needed to determine whether co-expression of specific MSC markers confers DP cells specific properties that could be used for the regeneration of human tissues, including the dental pulp, with standardized cell-based medicinal products

Exploring the complementarity of pancreatic ductal adenocarcinoma preclinical models

Purpose: Compare pancreatic ductal adenocarcinoma (PDAC), preclinical models, by their transcriptome and drug response landscapes to evaluate their complementarity. Experimental De-sign: Three paired PDAC preclinical models—patient‐derived xenografts (PDX), xenograft‐derived pancreatic organoids (XDPO) and xenograft‐derived primary cell cultures (XDPCC)—were derived from 20 patients and analyzed at the transcriptomic and chemosensitivity level. Transcriptomic characterization was performed using the basal‐like/classical subtyping and the PDAC molecular gradient (PAMG). Chemosensitivity for gemcitabine, irinotecan, 5‐fluorouracil and oxaliplatin was established and the associated biological pathways were determined using independent component analysis (ICA) on the transcriptome of each model. The selection criteria used to identify the different components was the chemosensitivity score (CSS) found for each drug in each model. Results: PDX was the most dispersed model whereas XDPO and XDPCC were mainly classical and basal-like, respectively. Chemosensitivity scoring determines that PDX and XDPO display a positive correlation for three out of four drugs tested, whereas PDX and XDPCC did not correlate. No match was observed for each tumor chemosensitivity in the different models. Finally, pathway analysis shows a significant association between PDX and XDPO for the chemosensitivity‐associated pathways and PDX and XDPCC for the chemoresistance‐associated pathways. Conclusions: Each PDAC preclinical model possesses a unique basal‐like/classical transcriptomic phenotype that strongly in-fluences their global chemosensitivity. Each preclinical model is imperfect but complementary, sug-gesting that a more representative approach of the clinical reality could be obtained by combining them. Translational Relevance: The identification of molecular signatures that underpin drug sensitivity to chemotherapy in PDAC remains clinically challenging. Importantly, the vast majority of studies using preclinical in vivo and in vitro models fail when transferred to patients in a clinical setting despite initially promising results. This study presents for the first time a comparison between three preclinical models directly derived from the same patients. We show that their applica-bility to preclinical studies should be considered with a complementary focus, avoiding tumor-based direct extrapolations, which might generate misleading conclusions and consequently the overlook of clinically relevant features.Fil: Hoare, Owen. Centre National de la Recherche Scientifique; FranciaFil: Fraunhoffer Navarro, Nicolas Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Elkaoutari, Abdessamad. Centre National de la Recherche Scientifique; FranciaFil: Gayet, Odile. Centre National de la Recherche Scientifique; FranciaFil: Bigonnet, Martin. Centre National de la Recherche Scientifique; FranciaFil: Roques, Julie. Centre National de la Recherche Scientifique; FranciaFil: Nicolle, Rémy. No especifíca;Fil: McGuckin, Colin. Cell Therapy Research Institute; FranciaFil: Forraz, Nico. Cell Therapy Research Institute; FranciaFil: Sohier, Emilie. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Tonon, Laurie. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Wajda, Pauline. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Boyault, Sandrine. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Attignon, Valéry. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Tabone, Luciana Belen. Le Centre Régional de Lutte Contre Le Cancer Léon Bérard; FranciaFil: Barbier, Sandrine. No especifíca;Fil: Mignard, Caroline. No especifíca;Fil: Duchamp, Olivier. No especifíca;Fil: Iovanna, Juan. Centre National de la Recherche Scientifique; FranciaFil: Dusetti, Nelson J.. Centre National de la Recherche Scientifique; Franci

Engineering of a Microscale Niche for Pancreatic Tumor Cells Using Bioactive Film Coatings Combined with 3D-Architectured Scaffolds

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The World’s First Acne Dysbiosis-like Model of Human 3D Ex Vivo Sebaceous Gland Colonized with <i>Cutibacterium acnes</i> and <i>Staphylococcus epidermidis</i>

Acne-prone skin is associated with dysbiosis involving Cutibacterium acnes (C. acnes) and Staphylococcus epidermidis (S. epidermidis) causing increased seborrhea in sebaceous glands (SG) and inflammation. Human primary sebocytes were cultivated using 1.106 UFC/mL C. acnes Type IA (facial acne, ATCC6919) and/or 1.105 UFC/mL S. epidermidis (unknown origin, ATCC12228) for 48 h in our SEB4GLN-optimized media without antibiotics. Bacteria and sebocytes were enumerated and assessed to determine their viability. Lipid production was imaged and quantified via Nile Red staining. SG with hair follicles were microdissected from healthy skin and cultured using 1.105 UFC/mL C. acnes Type 1A and/or 1.104 UFC/mL S. epidermidis (wild-type facial skin strain) through prior fixation and immunostaining for MC5R, C. acnes and nuclei (DAPI) via Z-stack confocal microscopy bioimaging (Leica SP5X & FIJI software, Version 2.9.0). C. acnes growth was not impacted when co-cultivated with sebocytes (2D) or SG (3D) models. Phylotype IA stimulated sebocyte lipid production, which had no impact on viability. The S. epidermidis reference strain overproliferated, inducing sebocyte mortality. For 3D SG model, culture conditions were optimized using a wild-type facial skin strain at a lower concentration, 1:10 ratio to C. acnes, reduced contact time, sequential inoculation and rinsing step. Bioimaging revealed strong C. acnes labeling in the active areas of the pilosebaceous unit. S. epidermidis formed biofilm, which was distributed across the SG via non-specific fluorescence imaging. We developed an innovative model of a sebaceous gland that mimics acne-prone skin with lipid overproduction and virulent phylotype IA C. acnes inoculation

Production of Human Dental Pulp Cells with a Medicinal Manufacturing Approach

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Topical Administration of <i>Lactiplantibacillus plantarum</i> (SkinDuo^TM) Serum Improves Anti-Acne Properties

The tailoring of the skin microbiome is challenging and is a research hotspot in the pathogenesis of immune-mediated inflammatory skin diseases such as acne. Commonly encountered preservatives used as functional ingredients have an impact on the skin microbiota and are known to inhibit the survival of skin commensal bacteria. The selected species is Lactiplantibacillus plantarum, formulated with natural enhancers for topical use (SkinDuoTM). Ex vivo human skin models were used as a test system to assess the strain viability which was then validated on healthy volunteers. SkinDuoTM showed increased viability over time for in vitro skin models and a stable viability of over 50% on healthy skin. The strain was tested on human primary sebocytes obtained from sebaceous gland rich areas of facial skin and inoculated with the most abundant bacteria from the skin microbiota. Results on human ex vivo sebaceous gland models with the virulent phylotype of Cutibacterium acnes and Staphylococcus epidermidis present a significant reduction in viability, lipid production, and anti-inflammatory markers. We have developed an innovative anti-acne serum with L. plantarum that mimics the over-production of lipids, anti-inflammatory properties, and improves acne-disease skin models. Based on these results, we suggest that SkinDuoTM may be introduced as an acne-mitigating agent

Topical Administration of Lactiplantibacillus plantarum (SkinDuoTM) Serum Improves Anti-Acne Properties

The tailoring of the skin microbiome is challenging and is a research hotspot in the pathogenesis of immune-mediated inflammatory skin diseases such as acne. Commonly encountered preservatives used as functional ingredients have an impact on the skin microbiota and are known to inhibit the survival of skin commensal bacteria. The selected species is Lactiplantibacillus plantarum, formulated with natural enhancers for topical use (SkinDuoTM). Ex vivo human skin models were used as a test system to assess the strain viability which was then validated on healthy volunteers. SkinDuoTM showed increased viability over time for in vitro skin models and a stable viability of over 50% on healthy skin. The strain was tested on human primary sebocytes obtained from sebaceous gland rich areas of facial skin and inoculated with the most abundant bacteria from the skin microbiota. Results on human ex vivo sebaceous gland models with the virulent phylotype of Cutibacterium acnes and Staphylococcus epidermidis present a significant reduction in viability, lipid production, and anti-inflammatory markers. We have developed an innovative anti-acne serum with L. plantarum that mimics the over-production of lipids, anti-inflammatory properties, and improves acne-disease skin models. Based on these results, we suggest that SkinDuoTM may be introduced as an acne-mitigating agent

Osteoblastic differentiation of Wharton jelly biopsy specimens and their mesenchymal stromal cells after serum-free culture

Cleft lip and cleft palate are increasingly being detected by prenatal ultrasound, which raises the opportunity of using the patient's own osteogenicity from umbilical cord mesenchymal cells for bony repair. The authors address the growth of the cells under a fully defined and regulated protocol.; Wharton jelly-derived mesenchymal stromal cells were isolated and expanded as a monolayer with defined serum-free medium. Osteoblastic differentiation was tested in the cells and in the entire Wharton jelly biopsy specimens. The serum-free-cultured cells were included in hydroxyapatite granule-fibrin constructs and, without predifferentiation, subcutaneously implanted into immunoincompetent mice.; Isolation and expansion of Wharton jelly-derived mesenchymal stromal cells were consistently successful under serum-free conditions, and the cells expressed standard mesenchymal stromal cell markers. The serum-free-cultivated cells produced a mineralized extracellular matrix under osteogenic differentiation, with a significant increase of osteoblastic lineage gene expression (Hox-A10 and Runx2) and an up-regulation of downstream osteogenic genes (OSX, OCN, ALPL, and BSP2). In vivo, they formed a dense matrix adjacent to the granules after 8 weeks, but no lamellar bone. serum-free-cultivated entire Wharton jelly biopsy specimens produced a mineralized extracellular matrix within the collagen matrix of the Wharton jelly.; The osteogenic differentiation potential of Wharton jelly-derived mesenchymal stromal cells was maintained under serum-free isolation and expansion techniques. The cells without predifferentiation form a dense collagen matrix but not bone in vivo. Moreover, entire Wharton jelly biopsy specimens showed periosteal-like mineralization under osteogenic differentiation, which offers new options for autologous bone tissue engineering, including cleft palate surgery

Characterization of Different Sources of Human MSCs Expanded in Serum-Free Conditions with Quantification of Chondrogenic Induction in 3D

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Immunophenotyping reveals the diversity of human dental pulp mesenchymal stromal cells in vivo and their evolution upon in vitro amplification

Mesenchymal stromal/stem cells (MSCs) from human dental pulp (DP) can be expanded in vitro for cell-based and regenerative dentistry therapeutic purposes. However, their heterogeneity may be a hurdle to the achievement of reproducible and predictable therapeutic outcomes. To get a better knowledge about this heterogeneity, we designed a flow cytometric strategy to analyze the phenotype of DP cells in vivo and upon in vitro expansion with stem cell markers. We focused on the CD31- cell population to exclude endothelial and leukocytic cells. Results showed that the in vivo CD31- DP cell population contained 1.4% of CD56+, 1.5% of CD146+, 2.4% of CD271+ and 6.3% of MSCA-1+ cells but very few Stro-1+ cells (≤1%). CD56+, CD146+, CD271+ and MSCA-1+ cell subpopulations expressed various levels of these markers. CD146+MSCA-1+, CD271+MSCA-1+ and CD146+CD271+ cells were the most abundant DP-MSC populations. Analysis of DP-MSCs expanded in vitro with a medicinal manufacturing approach showed that CD146 was expressed by about 50% of CD56+, CD271+, MSCA-1+ and Stro-1+ cells, and MSCA-1 by 15-30% of CD56+, CD146+, CD271+ and Stro-1+ cells. These ratios remained stable with passages. CD271 and Stro-1 were expressed by less than 1% of the expanded cell populations. Interestingly, the percentage of CD56+ cells strongly increased from P1 (25%) to P4 (80%) both in all sub-populations studied. CD146+CD56+, MSCA-1+CD56+ and CD146+MSCA-1+ cells were the most abundant DP-MSCs at the end of P4. These results established that DP-MSCs constitute a heterogeneous mixture of cells in pulp tissue in vivo and in culture, and that their phenotype is modified upon in vitro expansion. Further studies are needed to determine whether co-expression of specific MSC markers confers DP cells specific properties that could be used for the regeneration of human tissues, including the dental pulp, with standardized cell-based medicinal products