Current Issues and Regulations in Tendon Regeneration and Musculoskeletal Repair with Mesenchymal Stem Cells

Mesenchymal stem cells are multipotent stromal cells residing within the connective tissue of most organs. Their surface phenotype has been well described. Most commonly, mesenchymal stem cells demonstrate the ability to differentiate into mesenchymal tissues (bone, catailge, fat, etc...), however, under the proper conditions these cells can differentiate into epithelial cells and neuroectoderm derived lineages. Their developmental plasticity also depends on the ability of mesenchymal stem cells to alter the tissue microenvironment by secreting soluble factors, as well as their capacity for differentiation in tissue repair. It is the cell-matrix interaction which defines the tissue characteristics. The molecular and functional heterogeneity of this cell population may confound interpretation of their differentiation potential, but it is this heterogeneity that is believed to provide for their therapeutic efficacy. Stem cell therapies are an attractive therapeutic approach for soft tissues as they offer a vehicle for repair and regeneration at the end of a needle. The early introduction of stem cell treatments into the therapeutic armamentarium involves both commercial and non-commercial multidisciplinary partnerships and has occurred in a climate of regulatory reform, so not all the relevant information resides in the public domain, but early clinical studies have shown promising results. Against this backdrop, novel techniques and early results of a small series of tendon and musculotendinous junction interventions are being published and other ongoing studies are yet to report their results. The issue of ensuring governance of these novel technologies falls upon both the scientific community and the established licensing authorities

Stem Cells in Treatment of Coronary Heart Disease and Its Monitoring: Tissue Engineering and Clinical Evaluation

Cardiovascular and coronary heart diseases involve molecular and tissue level damage of blood vessels and heart. Coronary Heart Disease and heart failure are the leading cause of mortality worldwide. Stem cell transplantation is emerging as a new treatment option. Stem cells are capable to reach and settle down at damaged cardiac tissue. This stem cell option also repairs the myocardial infarction area in heart or vascular territories and ultimately reduces the infarct-related mortality. Non-invasive cardiovascular imaging monitors the real-time status of cardiovascular remodeling or differentiated stem cell autografting. Cardiac magnetic resonance imaging (MRI) and bioluminescence are robust non-invasive monitoring techniques to visualize cardiovascular structure changes due to myocardial dysfunction or restorative myocardial recovery. The present chapter highlights the sources, types, delivery methods of stem cells in cardiovascular treatment, advantages and current limitations of stem cell monitoring, scopes of ultra-high field cardiac 900 MHz MRI and bioluminescence methods applied in stem cell transplantation, to translate stem cell molecular events into clinical success and evaluation of rejuvenation rate with future perspectives. In conclusion, right choice of stem cells, pluripotent stem cell delivery, transplantation and real-time monitoring of stem cell trafficking enhances the stem cell therapeutic efficacy in cardiac engraftment and differentiation

Endogenous musculoskeletal tissue engineering - a focused perspective

Two major difficulties facing widespread clinical implementation of existing Tissue Engineering (TE) strategies for the treatment of musculoskeletal disorders are (1) the cost, space and time required for ex vivo culture of a patient’s autologous cells prior to re-implantation as part of a TE construct, and (2) the potential risks and availability constraints associated with transplanting exogenous (foreign) cells. These hurdles have led to recent interest in endogenous TE strategies, in which the regenerative potential of a patient’s own cells is harnessed to promote tissue regrowth without ex vivo cell culture. This article provides a focused perspective on key issues in the development of endogenous TE strategies, progress to date, and suggested future research directions toward endogenous repair and regeneration of musculoskeletal tissues and organs

Development of Bioartificial Myocardium Using Stem Cells and Nanobiotechnology Templates

Cell-based regenerative therapy is undergoing experimental and clinical trials in cardiology, in order to limit the consequences of decreased contractile function and compliance of damaged ventricles following myocardial infarction. Over 1000 patients have been treated worldwide with cell-based procedures for myocardial regeneration. Cellular cardiomyoplasty seems to reduce the size and fibrosis of infarct scars, limit adverse postischemic remodelling, and improve diastolic function. The development of a bioartificial myocardium is a new challenge; in this approach, tissue-engineered procedures are associated with cell therapy. Organ decellularization for bioscaffolds fabrication is a new investigated concept. Nanomaterials are emerging as the main candidates to ensure the achievement of a proper instructive cellular niche with good drug release/administration properties. Investigating the electrophysiological properties of bioartificial myocardium is the challenging objective of future research, associating a multielectrode network to provide electrical stimulation could improve the coupling of grafted cells and scaffolds with host cardiomyocytes. In summary, until now stem cell transplantation has not achieved clear hemodynamic benefits for myocardial diseases. Supported by relevant scientific background, the development of myocardial tissue engineering may constitute a new avenue and hope for the treatment of myocardial diseases

Stem and progenitor cell-based therapy in ischaemic heart disease: promise, uncertainties, and challenges

In the absence of effective endogenous repair mechanisms after cardiac injury, cell-based therapies have rapidly emerged as a potential novel therapeutic approach in ischaemic heart disease. After the initial characterization of putative endothelial progenitor cells and their potential to promote cardiac neovascularization and to attenuate ischaemic injury, a decade of intense research has examined several novel approaches to promote cardiac repair in adult life. A variety of adult stem and progenitor cells from different sources have been examined for their potential to promote cardiac repair and regeneration. Although early, small-scale clinical studies underscored the potential effects of cell-based therapy largely by using bone marrow (BM)-derived cells, subsequent randomized-controlled trials have revealed mixed results that might relate, at least in part, to differences in study design and techniques, e.g. differences in patient population, cell sources and preparation, and endpoint selection. Recent meta-analyses have supported the notion that administration of BM-derived cells may improve cardiac function on top of standard therapy. At this stage, further optimization of cell-based therapy is urgently needed, and finally, large-scale clinical trials are required to eventually proof its clinical efficacy with respect to outcomes, i.e. morbidity and mortality. Despite all promises, pending uncertainties and practical limitations attenuate the therapeutic use of stem/progenitor cells for ischaemic heart disease. To advance the field forward, several important aspects need to be addressed in carefully designed studies: comparative studies may allow to discriminate superior cell populations, timing, dosing, priming of cells, and delivery mode for different applications. In order to predict benefit, influencing factors need to be identified with the aim to focus resources and efforts. Local retention and fate of cells in the therapeutic target zone must be improved. Further understanding of regenerative mechanisms will enable optimization at all levels. In this context, cell priming, bionanotechnology, and tissue engineering are emerging tools and may merge into a combined biological approach of ischaemic tissue repai

Cardiomyogenic potentiality of somatic and stem cells when cultured in the three-dimensional peptide scaffold RAD16-I

Les malalties cardiovasculars són una de les majors causes de mortalitat a escala mundial. L’infart de miocardi és el principal responsable de les cardiopaties isquèmiques. La irrigació sanguínia al cor es veu bloquejada degut a una oclusió en un capil•lar sanguini provocant mort cel•lular massiva que genera una zona miocardíaca necròtica. En la última dècada, la medicina cardíaca regenerativa s’ha focalitzat en estratègies fonamentades en l’enginyeria de teixits i la teràpia cel•lular basada en cèl•lules mare. En aquest treball, hem caracteritzat el potencial cardíac de diferents tipus cel•lulars cultivats en bastides tri-dimensionals (3D) generades a partir de l’hidrogel peptídic RAD16-I. En primer lloc, hem estudiat l’adquisició de potencial mesenquimàtic de fibroblasts humans de dermis (hNDFs) en cultius 3D i la seva diferenciació subseqüent a llinatges adipogènic i cardiogènic. Únicament els hNDFs cultivats en hidrogels de RAD16-I adquireixen una potenciació mesenquimàtica. Les cèl•lules adopten espontàniament propietats semblants a les cèl•lules mare mesenquimàtiques mentre que la diferenciació a adipogènesis i cardiogènesis requereix medi d’inducció. En segon lloc, hem comparat el grau de diferenciació cardíaca de cèl•lules mare humanes pluripotents induïdes (hiPSCs) cultivades en ambients 2D versus 3D i hem avaluat l’efecte de l’àcid ascòrbic (AA) en el procés. En el nostre treball i com ja s’havia demostrat en publicacions prèvies, l’AA va resultar accelerar i millorar la diferenciació cardíaca de hiPSCs en cultius 2D. A més, els resultats presentats suggereixen que les hiPSCs cultivades en 3D augmenten el seu grau de diferenciació i adquireixen un potencial cardiogènic 105 vegades més elevat que en els cultius 2D. En tercer lloc, hem dissenyat un pegat cardíac basat en cultius 3D de cèl•lules adultes porcines progenitores del teixit adipós del mediastí (pMATPCs) injectats en matrius naturals (pericardi humà descel•lularitzat). Hem implantat la bio-pròtesis miocardíaca in vivo i hem determinat que la bio-bastida afavoreix la migració cel•lular i la regeneració de la zona infartada en el model porcí. En conclusió, hem analitzat el potencial cardiogènic de cèl•lules adultes somàtiques (hNDFs), cèl•lules mare adultes (pMATPCs) i cèl•lules mare pluripotents (hiPSCs) en cultius 3D basats en hidrogels de RAD16-I per a futures aplicacions en el tractament de malalties cardíaques.Las enfermedades cardiovasculares son una de las mayores causas de mortalidad a escala mundial. El infarto de miocardio es el principal responsable de las cardiopatías isquémicas. La irrigación sanguínea al corazón se ve bloqueada debido a una oclusión en un capilar sanguíneo provocando muerte celular masiva que genera una zona miocárdica necrótica. En la última década, la medicina cardíaca regenerativa se ha focalizado en estrategias fundamentadas en la ingeniería de tejidos y la terapia celular basada en células madre. Es este trabajo, hemos caracterizado el potencial cardíaco de distintos tipos celulares cultivados en andamios tridimensionales (3D) generados a partir del hidrogel peptídico RAD16-I. En primer lugar, hemos estudiado la adquisición de potencial mesenquimático de fibroblastos humanos de dermis (hNDFs) en cultivos 3D y su diferenciación subsecuente a linajes adipogénico y cardiogénico. Únicamente los hNDFs cultivados en hidrogeles de RAD16-I adquieren una potenciación mesenquimática. Las células adoptan espontáneamente propiedades parecidas a las células madre mesenquimáticas mientras que la diferenciación a adipogénesis y cardiogénesis requiere medio de inducción. En segundo lugar, hemos comparado el grado de diferenciación cardíaca de células madre humanas pluripotentes inducidas (hiPSCs) cultivadas en ambientes 2D versus 3D y hemos evaluado el efecto del ácido ascórbico (AA) en el proceso. En nuestro trabajo y como ya se había demostrado en publicaciones previas, el AA resultó acelerar y mejorar la diferenciación cardíaca de hiPSCs en cultivos 2D. A demás, los resultados presentados sugieren que las hiPSCs cultivadas en 3D aumentan su grado de diferenciación y adquieren un potencial cardiogénico 105 veces más elevado que en los cultivos 2D. En tercer lugar, hemos diseñado un parche cardíaco basado en cultivos 3D de células adultas porcinas progenitoras del tejido adiposo del mediastino (pMATPCs) inyectados en matrices naturales (pericardio humano descelularizado). Hemos implantado la bio-prótesis miocárdica in vivo y hemos determinado que el bio-andamio favorece la migración celular y la regeneración de la zona infartada en el modelo porcino. En conclusión, hemos analizado el potencial cardiogénico de células adultas somáticas (hNDFs), células madre adultas (pMATPCs) y células madre pluripotentes (hiPSCs) en cultivos 3D basados en hidrogeles de RAD16-I para futuras aplicaciones en el tratamiento de enfermedades cardíacas.Cardiac failure is the primary cause of mortality throughout the world. One of the leading causes of heart failure is myocardial infarction, which results from a reduced flow of blood to a part of the heart. This leads to cardiomyocyte death and myocardial necrosis. In the past decade, various strategies for cardiac reparative medicine have been investigated, from tissue engineering to stem cell-based therapy. Herein, we characterized the cardiac potential of different cell types cultured in three-dimensional (3D) scaffolds based on the peptide hydrogel RAD16-I. Firstly, we studied the mesenchymal potential acquisition of human Normal Dermal Fibroblasts (hNDFs) in 3D cultures and further commitment into adipogenic and cardiogenic lineages. We suggest that only hNDFs cultured in RAD16-I hydrogels undergo a mesenchymal potentiation. Cells spontaneously acquired mesenchymal stem cell-like properties whereas they required induction media to differentiate into adipogenic- and cardiogenic-like lineages. Secondly, we compared the degree of cardiac commitment of human induced Pluripotent Stem Cells (hiPSCs) when cultured in 2D versus 3D and the effect of ascorbic acid (AA), which has been proven to promote cardiac differentiation, on the process. In fact, AA seemed to accelerate and improve the cardiac commitment of hiPSCs in 2D cultures. Results suggested that hiPSCs in 3D cultures displayed an increased level of differentiation and acquired 105-fold more cardiogenic potential than cells cultured in 2D. Thirdly, we designed a cardiac patch based on 3D cultures of adult porcine Mediastinal Adipose Tissue Progenitor Cells (pMATPCs) injected into natural matrices (decellularized human pericardium). We implanted the myocardial bioprosthesis in vivo and determined that the bioscaffold supported cell migration and regeneration into the infarcted area in swine. In summary, we studied the cardiogenic potential of adult somatic cells (hNDFs), adult stem cells (pMATPCs) and pluripotent stem cells (hiPSCs) in 3D cultures based on RAD16-I hydrogels for potential future applications in the treatment of heart disease

Disclosing CCBE1 role in Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiovascular diseases (CVD) are the leading cause of death worldwide. Within CVDs, myocardial infarction (MI) is associated with a massive and irreversible loss of cardiomyocytes (CM). An in-depth comprehension of key cellular mechanisms and molecules involved in cardiogenesis is fundamental to improve cardiac therapies by exposing novel therapeutic targets. CCBE1, a collagen and calcium-EGF biding domain 1 protein, was identified to be expressed in mouse heart precursors. Mutations in CCBE1 have been associated with Hennekam syndrome, which is characterized by abnormal lymphatic system and congenital heart defects. However, the CCBE1 functional role in cardiac specification is still unknown. Therefore, the main aim of this thesis was to unveil CCBE1 role in CM and Endothelial cells (EC) specification. For this purpose, a modified hiPSC line displaying the CRISPR interference technology (CRISPRi) was used to selectively knockdown (KD) CCBE1 gene expression along CM and EC differentiation. We showed that CCBE1 downregulation did not affect hiPSCs growth, morphology and stemness. Nonetheless, a significant reduction on gene expression of cardiac troponin T2 gene (TNNT2) and lower gene expression ratios of cardiac troponin I isoforms (TNNI3:TNNI1) and myosin heavy chains (MYH7:MYH6) were detected in CMs derived from CRISPRi-CCBE1 KD cell line at day 15. Ultrastructural changes were also observed in this condition, CMs presented lower sarcomere length and alignment, indicating a more immature state. In contrast, EC differentiation was not affected by CCBE1 KD, with no impact on EC morphology or gene expression levels. Therefore, CCBE1 seems to have a key role on CM specification and maturation. Moreover, we successfully selected hiPSC clonal populations with higher level of CCBE1 KD for future studies. This work may contribute with new insights towards the development of CCBE1-mediated therapeutic strategies for cardiac regenerative medicine

Tissue Engineering Strategies for Myocardial Regeneration: Acellular Versus Cellular Scaffolds?

Heart disease remains one of the leading causes of death in industrialized nations with myocardial infarction (MI) contributing to at least one fifth of the reported deaths. The hypoxic environment eventually leads to cellular death and scar tissue formation. The scar tissue that forms is not mechanically functional and often leads to myocardial remodeling and eventual heart failure. Tissue engineering and regenerative medicine principles provide an alternative approach to restoring myocardial function by designing constructs that will restore the mechanical function of the heart. In this review, we will describe the cellular events that take place after an MI and describe current treatments. We will also describe how biomaterials, alone or in combination with a cellular component, have been used to engineer suitable myocardium replacement constructs and how new advanced culture systems will be required to achieve clinical success