L'eterogeneità dei fibroblasti dermici come fattore in grado di condizionare l'efficacia della riprogrammazione cellulare. Dermal fibroblasts heterogeneity as a major factor influencing the efficiency of cell reprogramming.

Fibroblasts are a heterogeneous cell population as testified by the absence of specific markers and by their documented retention of positional memory. Fibroblasts have recently gained much attention as they are the adult somatic cell most commonly used for reprogramming to induced pluripotent stem cells (iPSCs). iPSCs are somatic cells genetically reprogrammed to a pluripotent state that makes them resemble embryonic stem cells (ESCs). iPSCs are a very promising tool for regenerative medicine because of their autologous origin and their ability to differentiate towards cell of all three germ layers. However, the iPSCs reprogramming technology is still affected by variable and low efficiency. We hypothesize that the heterogeneity of fibroblasts may play a role in determining cell reprogramming. To test our hypothesis, we isolated and cultured human dermal fibroblasts (HDFs) from five different anatomic sites (abdomen, breast, neck, thigh and arm) and performed a comparative study analyzing their morphology, proliferation, apoptosis and ability to migrate. Further, we evaluated whether the expression of mesenchymal and epithelial markers, the ability to differentiate and release growth factors in culture medium varied among fibroblasts from different anatomic sites. Our results revealed that, although fibroblasts from different anatomic sites had similar morphology, all other features varied according to the region from which they were isolated. Furthermore, we observed similarity between fibroblasts and mesenchymal stem cells (MSCs) that also varied among HDFs populations. A low proliferation index, the release of specific growth factors, the expression of mesenchymal markers and the promptness to differentiate towards mesodermal lineages, might improve the efficiency of reprogramming. On these bases, abdomen dermal fibroblasts might represent the ideal candidates for reprogramming; since they have a low proliferation index, express high amount of mesenchymal markers, differentiate more precociously and release growth factors that improve the efficiency of reprogramming. Further the high expression of mesenchymal markers and their ability to differentiate more precociously suggest that fibroblasts isolated from abdomen skin could represent a less differentiated phenotype, more similar to MSCs than the other population of fibroblasts and probably easier to reprogram. Therefore, our data offer novel perspectives in the field of cell reprogramming, suggesting that the choice of the dermal population of fibroblasts might influence the efficiency of reprogramming itself

Metabolic Reprogramming of Cancer Associated Fibroblasts: The Slavery of Stromal Fibroblasts

Cancer associated fibroblasts (CAFs) are the main stromal cell type of solid tumour microenvironment and undergo an activation process associated with secretion of growth factors, cytokines, and paracrine interactions. One of the important features of solid tumours is the metabolic reprogramming that leads to changes of bioenergetics and biosynthesis in both tumour cells and CAFs. In particular, CAFs follow the evolution of tumour disease and acquire a catabolic phenotype: in tumour tissues, cancer cells and tumour microenvironment form a network where the crosstalk between cancer cells and CAFs is associated with cell metabolic reprogramming that contributes to CAFs activation, cancer growth, and progression and evasion from cancer therapies. In this regard, the study of CAFs metabolic reprogramming could contribute to better understand their activation process, the interaction between stroma, and cancer cells and could offer innovative tools for the development of new therapeutic strategies able to eradicate the protumorigenic activity of CAFs. Therefore, this review focuses on CAFs metabolic reprogramming associated with both differentiation process and cancer and stromal cells crosstalk. Finally, therapeutic responses and potential anticancer strategies targeting CAFs metabolic reprogramming are reviewed

Positional memory of fibroblasts may affect efficiency of iPSC reprogramming

Induced Pluripotent Stem cells (iPSC) are pluripotent stem cells reprogrammed from adult somatic cells. Although iPSC hold great potential for applications in regenerative medicine, technical problems, mostly related to the low efficiency of reprogramming, are yet to be solved. Since the most used cells for iPSC reprogramming are skin fibroblasts (FB), and since FB preserve positional memory, we hypothesize that the anatomic origin of FB might influence iPSC reprogramming.We isolated FB from skin of five different sites (neck, arm, thigh, breast, abdomen) of 13 patients undergoing plastic surgery or from heart wall or ascending aorta wall of the explanted heart of 3 patients receiving heart transplantation. FB from different anatomic sites and control FB from neonatal foreskin, were cultured for one week to evaluate morphology, proliferation rate and proneness to apoptosis. Additionally, expression of vimentin, cadherin, smooth muscle actin and Factor VIII was investigated to exclude the presence of other cell types. Transcriptome analysis including genes involved in stemness maintenance, embryogenesis, cell growth, activation and development, was performed by real-time PCR. Despite the similar morphology of FB from different sites, and immunopositivity for vimentin, along with the absence of other cell type markers, FB isolated from abdomen and heart had 1.5-fold higher doubling time, while FB from heart, abdomen and breast were less susceptible to apoptosis. Intriguingly, Real-Time PCR revealed that in abdomen, breast, neck, arm and heart FB genes involved in cell growth, development, proliferation, and migration, as TM4SF1, GPC4, CSPG2, DDIT4, ID1 were up-regulated, while genes regulating embryogenesis and tissue morphogenesis, like VCAN, FN1, HOXA5, CD49a were up-regulated in FB isolated from abdomen, arm and heart. However, all FBs had transcripts of markers of Mesenchymal Stem Cells (MSC), as CD105 and CD90. Our results provide evidence that human adult FB from different sites have different genetic program. Therefore, FB may respond to reprogram technology in different manner, thus affecting reprogramming efficiency. While offering novel perspective of the reprogramming technology, our study also demonstrates that abdomen and breast FB share cardiac genetic signature of cardiac FB while expressing markers of MSC and they might represent the ideal cell for cardiac reprogramming

From Cover to Core: Acellular Human Dermis for the Regeneration of Human Heart

Elasticity of myocardium is mostly due to elasticity of cardiomyocytes and is essential for cardiomyocyte alignment and differentiation. Cardiac decellularized ECM (d-ECM) is emerging as natural scaffold to promote and support myocardial regeneration. It is noteworthy that cardiac d-ECM is obtained through complete removal of cardiomyocytes with loss of elasticity. We hypothesize that decellularized skin might be an easily accessible, viable alternative for myocardium regeneration, as decellularization is unlikely to cause loss of skin elasticity, provided by elastic fibers rather than by resident cells. Skin fragments from patients undergoing plastic surgery were decellularized through novel simple and fast protocol. Decellularized Human Skin (d-HuSk) obtained was assayed in quantitative dye-binding method to measure content of elastin, while elastin distribution was evaluated on histological sections by Paraldehyde Fuchsin Gomori and Weigert Van Gieson stainings. d-Husk was then sectioned and used as scaffold to prepare three-dimensional culture of cardiac primitive cells (CPCs). Then, survival and ability of CPC cultured on d-HuSk to differentiate towards cardiac myocytes was evaluated at gene and protein level. Histological and quantitative analysis provided evidence of effective decellularization, preserved tissue architecture and retention of elastin. CPCs engrafted onto d-Husk, survived, and retained expression of markers specific for cardiac myocytes at gene and protein level.Our study provides compelling evidence that common signals act in cardiac and skin microenvironment to maintain CPC ability to differentiate towards cardiac muscle and that skin holds promise as an alternate biological scaffold for cardiovascular regenerative medicine

Optimization of human heart decellularization method for cardiac regenerative medicine

Extracellular matrix (ECM) is an intricate mesh of collagenous and non-collagenous proteins, whose presence and amount vary according to type of tissue. ECM drew the attention of regenerative medicine scientists as natural scaffold suitable for stem cell delivery into damaged tissues. Although a multitude of protocols and combinations of chemical agents and physical methods have been tested and proved effective in the decellularization of human heart, none of the ones tried in our setting fulfilled the goal of obtaining a structurally preserved cardiac decellularized ECM (d-ECM). While testing already described procedures, we made several adjustments that led to the development of a novel, simpler and robust protocol to decellularize adult human heart. Specifically, we decellularized cardiac samples of the free wall of both ventricles of adult human hearts scaled down to fit into embedding cassettes used to avoid stirring stress and preserve structure. To shorten the procedure, a combination of SDS, Triton X-100 and antibiotics was used in simple and fast two-step protocol. After decellularization, d-ECM was fixed and processed for histological study or snap-frozen for molecular biology analysis or cytocompatibility test in vitro. Histochemistry and immunoistochemistry confirmed the absence of nuclei and the preservation of architecture and composition of d-ECM. Further, while DNA content in d-ECM was well below accepted standards, sGAG, elastin and growth factors were retained and d-ECM scaffolds supported cardiac primitive cell engraftment and survival in vitro. Hence, according to our evidence, our protocol is simple, fast, effective and is worth improving for clinical translation

Positional memory of fibroblast may affect efficiency of iPSC reprogramming.

Induced Pluripotent Stem cells (iPSC) are pluripotent stem cells reprogrammed from adult somatic cells. Although iPSC hold great potential for applications in regenera-tive medicine, technical problems, mostly related to the low efficiency of reprogram-ming, are yet to be solved. Since the most used cells for iPSC reprogramming are skin fibroblasts (FB), and since FB preserve positional memory, we hypothesize that the anatomic origin of FB might influence iPSC reprogramming. We isolated FB from skin of five different sites (neck, arm, thigh, breast, abdomen) of 13 patients undergoing plastic surgery or from heart wall or ascending aorta wall of the explanted heart of 3 patients receiving heart transplantation. FB from different anatomic sites and control FB from neonatal foreskin, were cultured for one week to evaluate morphology, proliferation rate and proneness to apoptosis. Additionally, ex-pression of vimentin, cadherin, smooth muscle actin and Factor VIII was investigated to exclude the presence of other cell types. Transcriptome analysis including genes involved in stemness maintenance, embryogenesis, cell growth, activation and development, was performed by real-time PCR. Despite the similar morphology of FB from different sites, and immunopositivity for vimentin, along with the absence of other cell type markers, FB isolated from abdomen and heart had 1.5-fold higher doubling time, while FB from heart, abdomen and breast were less susceptible to apoptosis. Intriguingly, Real-Time PCR revealed that in abdomen, breast, neck, arm and heart FB genes involved in cell growth, development, proliferation, and migration, as TM4SF1, GPC4, CSPG2, DDIT4, ID1 were up-regulated, while genes regulating embryogenesis and tissue morphogenesis, like VCAN, FN1, HOXA5, CD49a were up-regulated in FB isolated from abdomen, arm and heart. However, all FBs had transcripts of markers of Mesenchymal Stem Cells (MSC), as CD105 and CD90. Our results provide evidence that human adult FB from different sites have different genetic program. Therefore, FB may respond to reprogram technology in different manner, thus affecting reprogramming efficiency. While offering novel perspective of the reprogramming technology, our study also demonstrates that abdomen and breast FB share cardiac genetic signature of cardiac FB while expressing markers of MSC and they might represent the ideal cell for cardiac reprogramming

Decellularized human skin as biological scaffold for cardiovascular repair and regeneration

INTRODUCTION Skin shares properties of elasticity with muscular tissue. Since elasticity is mostly conferred by muscle cells or elastic fibers, after decellularization the removal of muscle cells causes in decellularized muscles loss of such property, while decellularized skin retains elasticity as skin ECM is rich in elastic fibers that are retained after decellularization. Additionally, mechanic properties are fundamental to ensure myocyte differentiation1 and alignment in myocardium. EXPERIMENTAL METHODS We developed a fast and efficient protocol of decellularization for human skin using skin fragments from patients undergoing plastic surgery. After decellularization, content of elastin was quantified by quantitative dye-binding method. Additionally elastin content and distribution was evaluated on histological sections by Paraldehyde Fuchsin Gomori and Weigert Van Gieson stainings. Decellularized Human SKin (d-HuSk) obtained was then sectioned into 600um thick sections and used as scaffold to prepare three-dimensional culture of cardiac primitive cells (CPCs). We evaluated, then, CPC survival and ability to differentiate, in vitro, towards cardiomyocytes at gene and protein level when cultured on d-HuSk. RESULTS AND DISCUSSION Decellularization procedure yielded the acellular extracellular matrix (ECM) with preserved tissue architecture, named d-HuSk. Importantly, histological and quantitative analysis clearly showed the retention of elastic fibers by d-HuSk. CPCs seeded on d-Husk engrafted and survived, and their ability to differentiate towards cardiomyocytes was not lost, as shown by preserved expression of markers specific for cardiac muscle cells, both at protein and gene level. Such results suggest that common signals and properties act both in cardiac and skin microenvironment, making skin a potential powerful and off-the-shelf biological scaffold for cardiovascular regenerative medicine. CONCLUSION Although emerging from an in vitro study, the evidence that progenitors of cardiac muscle lineage retain the ability to differentiate on biological scaffold obtained from different, more easily accessible, anatomic site, represents an important advance in cardiovascular regenerative medicine. Specifically, d-HuSk is an alternate biological scaffold that overcomes problems related to the preparation of myocardial biological scaffolds

From Cover to Core: Acellular Human Dermis for the Regeneration of Human Heart

Elasticity of myocardium is mostly due to elasticity of cardiomyocytes and is essential for cardiomyocyte alignment and differentiation. Cardiac decellularized ECM (d-ECM) is emerging as natural scaffold to promote and support myocardial regeneration. It is noteworthy that cardiac d-ECM is obtained through complete removal of cardiomyocytes with loss of elasticity. We hypothesize that decellularized skin might be an easily accessible, viable alternative for myocardium regeneration, as decellularization is unlikely to cause loss of skin elasticity, provided by elastic fibers rather than by resident cells. Skin fragments from patients undergoing plastic surgery were decellularized through novel simple and fast protocol. Decellularized Human Skin (d-HuSk) obtained was assayed in quantitative dye-binding method to measure content of elastin, while elastin distribution was evaluated on histological sections by Paraldehyde Fuchsin Gomori and Weigert Van Gieson stainings. d-Husk was then sectioned and used as scaffold to prepare three-dimensional culture of cardiac primitive cells (CPCs). Then, survival and ability of CPC cultured on d-HuSk to differentiate towards cardiac myocytes was evaluated at gene and protein level. Histological and quantitative analysis provided evidence of effective decellularization, preserved tissue architecture and retention of elastin. CPCs engrafted onto d-Husk, survived, and retained expression of markers specific for cardiac myocytes at gene and protein level.Our study provides compelling evidence that common signals act in cardiac and skin microenvironment to maintain CPC ability to differentiate towards cardiac muscle and that skin holds promise as an alternate biological scaffold for cardiovascular regenerative medicine