Basic and translational research on lung biology has discovered multiple progenitor cell types, specialized or facultative, responsible for turnover, renewal, and repair. Isolation of populations of resident lung progenitor cells (LPCs) has been described by multiple protocols, and some have been successfully applied to healthy human lung tissue. We aimed at understanding how different cell culture conditions may affect, in vitro, the phenotype of LPCs to create an ideal niche‐like microenvironment. The influence of different substrates (i.e., fibronectin, gelatin, laminin) and the impact of a three‐dimensional/two‐dimensional (3D/2D) culture switch on the biology of LPCs isolated as lung spheroids (LSs) from normal adult human lung biopsy specimens were investigated. We applied a spheroid culture system as the selective/inductive step for progenitor cell culture, as described in many biological systems. The data showed a niche‐like proepithelial microenvironment inside the LS, highly sensitive to the 3D culture system and significantly affecting the phenotype of adult LPCs more than culture substrate. LSs favor epithelial phenotypes and LPC maintenance and contain cells more responsive to specific commitment stimuli than 2D monolayer cultures, while secreting a distinctive set of paracrine factors. We have shown for the first time, to our knowledge, how culture as 3D LSs can affect LPC epithelial phenotype and produce strong paracrine signals with a distinctive secretomic profile compared with 2D monolayer conditions. These findings suggest novel approaches to maintain ex vivo LPCs for basic and translational studies. Stem Cells Translational Medicine 2017;6:767–777

Human resident cardiac progenitor cells (CPCs) isolated as cardiosphere-derived cells (CDCs) are under clinical evaluation as a therapeutic product for cardiac regenerative medicine. Unfortunately, limited engraftment and differentiation potential of transplanted cells significantly hamper therapeutic success. Moreover, maladaptive remodelling of the extracellular matrix (ECM) during heart failure progression provides impaired biological and mechanical signals to cardiac cells, including CPCs. In this study, we aimed at investigating the differential effect on the phenotype of human CDCs of cardiac fibroblast-derived ECM substrates from healthy or diseased hearts, named, respectively, normal or pathological cardiogel (CG-N/P). After 7 days of culture, results show increased levels of cardiogenic gene expression (NKX2.5, CX43) on both decellularized cardiogels compared to control, while the proportion and staining patterns of GATA4, OCT4, NKX2.5, ACTA1, VIM, and CD90-positive CPCs were not affected, as assessed by immunofluorescence microscopy and flow cytometry analyses. Nonetheless, CDCs cultured on CG-N secreted significantly higher levels of osteopontin, FGF6, FGF7, NT-3, IGFBP4, and TIMP-2 compared to those cultured on CG-P, suggesting overall a reduced trophic and antiremodelling paracrine profile of CDCs when in contact with ECM from pathological cardiac fibroblasts. These results provide novel insights into the bidirectional interplay between cardiac ECM and CPCs, potentially affecting CPC biology and regenerative potential.

Several biological mechanisms of action have been described which can explain the multiple therapeutic effects of CPC treatment observed on cardiac function and remodelling. These mechanisms span from direct cardiovascular differentiation, through induction of resident progenitor proliferation, to paracrine effects on cardiac and non-cardiac cells mediated by exosomes and non-coding RNAs. All the reported mechanisms of action support an integrated view including cardiomyogenesis, cardioprotection, and anti-fibrotic effects. Moreover, future developments of CPC therapy approaches may support cell-free strategies, exploiting effective pleiotropic cell-derived products, such as exosomes.

Cardiac adverse remodeling is characterized by biological changes that affect the composition and architecture of the extracellular matrix (ECM). The consequently disrupted signaling can interfere with the balance between cardiogenic and pro-fibrotic phenotype of resident cardiac stromal primitive cells (CPCs). The latter are important players in cardiac homeostasis and can be exploited as therapeutic cells in regenerative medicine. Our aim was to compare the effects of human decellularized native ECM from normal (dECM-NH) or failing hearts (dECM-PH) on human CPCs. CPCs were cultured on dECM sections and characterized for gene expression, immunofluorescence, and paracrine profiles. When cultured on dECM-NH, CPCs significantly upregulated cardiac commitment markers (CX43, NKX2.5), cardioprotective cytokines (bFGF, HGF), and the angiogenesis mediator, NO. When seeded on dECM-PH, instead, CPCs upregulated pro-remodeling cytokines (IGF-2, PDGF-AA, TGF-β) and the oxidative stress molecule H2O2. Interestingly, culture on dECM-PH was associated with impaired paracrine support to angiogenesis, and increased expression of the vascular endothelial growth factor (VEGF)-sequestering decoy isoform of the KDR/VEGFR2 receptor. Our results suggest that resident CPCs exposed to the pathological microenvironment of remodeling ECM partially lose their paracrine angiogenic properties and release more pro-fibrotic cytokines. These observations shed novel insights on the crosstalk between ECM and stromal CPCs, suggesting also a cautious use of non-healthy decellularized myocardium for cardiac tissue engineering approaches.

The increased knowledge in cell signals and stem cell differentiation, together with the development of new technologies, such as 3D bioprinting, has made the generation of artificial tissues more feasible for in vitro studies and in vivo applications. In the human body, cell fate, function, and survival are determined by the microenvironment, a rich and complex network composed of extracellular matrix (ECM), different cell types, and soluble factors. They all interconnect and communicate, receiving and sending signals, modulating and responding to cues. In the cardiovascular field, the culture of stem cells in vitro and their differentiation into cardiac phenotypes is well established, although differentiated cardiomyocytes often lack the functional maturation and structural organization typical of the adult myocardium. The recreation of an artificial microenvironment as similar as possible to the native tissue, though, has been shown to partly overcome these limitations, and can be obtained through the proper combination of ECM molecules, different cell types, bioavailability of growth factors (GFs), as well as appropriate mechanical and geometrical stimuli. This review will focus on the role of the ECM in the regulation of cardiac differentiation, will provide new insights on the role of supporting cells in the generation of 3D artificial tissues, and will also present a selection of the latest approaches to recreate a cardiac microenvironment in vitro through 3D bioprinting approaches.