The longitudinal monitoring for the myocardium

The imaging techniques , in suitable parameters , provide the connection between the cell therapies and the clinical procedure . The longitudinal monitoring provides information about the functioning mechanism of the reprogrammed cells in the in vivo environment . The review presents the imaging techniques that are able to explain the cause for the limited restoration process of the cell therapies in the cardiac tissue .

The human body contains approximately 3 , 7 × 10¹³ cells . The heart contains 6 × 10⁹ cells , in 300 grams ( figure 1 ) . The damage of the heart does not lead automatically to the regeneration of the full functional capacity . The scar formation and the inflammatory signals may not be enough to have a full regeneration of the cardiac tissue . The regenerative medicine enhances the healing mechanisms of the body with the cell therapy .

Figure 1 . The number of cells in the parts of the human body .
Copyright ( 2014 ) Katie Vicari / Nature Publishing Group

The minimum number of injected cells that is detectable by the imaging techniques is in the range of 10⁴ - 10⁶ cells . The heart contains 2 × 10⁷ cells in one gram of tissue , in the left ventricle . The fused positron emission tomography ( PET ) - computed tomography ( CT ) image of the porcine heart is an example for the technique ( figure 2 ) . The long arrows point to the trajectory of the thoracotomy . T is the inserted tube . The short arrows point to the 10⁸ human mesenchymal stem cells , injected in the left ventricle ( LV ) . The %ID / g is the percentage of the uptake by the injected human cells in one gram of porcine cardiac tissue .

Figure 2 . The PET - CT imaging of the expression for the reporter gene in the porcine myocardium .
Copyright ( 2009 ) RSNA

The results of a porcine model for the myocardial infarction identifies as the time for the maximum proliferation at an interval of 33 - 35 days after the injection of the mesenchymal stem cells in the myocardium .

The injected cells are not tracked in vivo if they are not labelled in vitro . The nanoparticles , as the exogeneous label , provide a strong contrast in a short acquisition time , but it is unreliable in the long - term monitoring . The incorporation process of reporter genes labels indirectly the target cell . The reporter gene in the nucleus creates reporter proteins secreted in the cytoplasm . The imaging technique detects the reporter proteins . The ideal cell label stays inside the target cells for a long time , is non - toxic , is in a concentration that is stoichiometric related to the number of target cells , and clears the system rapidly after the apoptosis of the target cells .

The challenges of the imaging techniques are the label dilution , and the cell tracking . The label dilution should remain inside the tracked cells until the apoptosis , but sometimes the dilution transfers to the host cells . The long - term tracking of the implanted cells through the imaging techniques has the challenge to quantify the number of cells from the initial implantation procedure that are alive , and the number of cells that proliferate , in the spatial and the temporal dimension .

The infarcted myocardium is trully regenerated with the aid from the cell therapies when the viable cardiac tissue in the infarct area has the volume increased , the fiber architecture is integer in structure , and the regenerated myocardium has synchronous contraction with the host .

References :

Naumova A . V . , Modo M . , Moore A . , Murry C . E . , Frank J . A . ( 2014 ) “ Clinical imaging in regenerative medicine ” , Nature Biotechnology , 32 ( 8 ) , 804 - 818 .

Willmann J . K . , Paulmurugan R . , Rodriguez - Porcel M . , Stein W . , Brinton T . J . , Connolly A . J . , Nielsen C . H . , Lutz A . M . , Lyons J . , Ikeno F . , Suzuki Y . , Rosenberg J . , Chen I . Y . , Wu J . C . , Yeung A . C . , Yock P . , Robbins R . C . , Gambhir S . S . ( 2009 ) “ Imaging gene expression in human mesenchymal stem cells : from small to large animals ” , Radiology - Radiological Society of North America , 252 ( 1 ) , 117 - 127 .

Hydrogels in regenerative medicine

The muscle tissue of the heart is remodelled following the myocardial infarction . The ventricular wall is thinning and the chamber dilates . The adult cardiomyocytes regenerate at a lower rate than the heart requires to function at the required capacity , without any treatment . The clinical strategies are to either use synthetic implants , or to deliver cells directly into the tissue . A solution to avoid their shortcomings is to use hydrophilic hydrogels as a three dimensional structure to deliver cells into the body . The review in Nature presents the hydrogels that demonstrated the ability to enhance vascularisation , or to promote the differentiation of the delivered stem cells .

The mechanical properties of the synthetic hydrogels ( Poly – ethylene glycol , Poly – N – isopropylacrylamide , Poly - 2 – hydroxyethyl methacrylate ) have the ability to control the behaviour of the cells in the damaged area of the myocardium . The NIPAAm - co - AAc - co – HEMAPTMC is a thermosensitive hydrogel that reduces the dimensions of the dilated left ventricle , while implanted in the rat with myocardial infarction . Figure 1 presents the rat myocardium eight weeks after the implantation of the hydrogel . Natural hydrogels ( Matrigel , Fibrin , hydrogel from porcine myocardial extracellular matrix ) increase the cell viability .

Figure 1 . The morphology and the experimental outcomes of the NIPAAm - co - AAc - co – HEMAPTMC thermosensitive hydrogel . ( a ) The composition of the hydrogel . ( b ) The elastic properties of the hydrogel . ( c ) The control experiment with phosphate buffered saline ( PBS ) injection . ( d ) The rat heart after hydrogel injection . ( e ) The histology analysis with hematoxylin / eosin ( H & E ) of the control experiment . ( f ) The H & E histology analysis of the rat cardiac tissue with the injected hydrogel . ( g ) Immunohistochemistry for the tissue grown into the injected hydrogel . Copyright ( 2014 ) Nature Publishing Group .

The hydrogels that demonstrate elastic behaviour while exposed to mechanical cyclic loading have the potential to repair the cardiac tissue . The hybrid of resilin – like polypeptide and Poly – ethylene glycol promotes cell differentiation after seven days of culture . The properties of Methacrylated tropoelastin ( MeTro ) are summarised in figure 2 . The micropatterned surface promotes the alignment and maturation for the cardiac cells , in the day 8 of the experiment. The frequency of the spontaneous beating rates of the cardiomyocytes was measured during two weeks of culture .

Figure 2 . The markers expressed by the primary rat cardiomyocytes . ( a ) The troponin I ( green ) and the nuclei ( blue ) on the surface patterned with The Methacrylated Tropoelastin ( MeTro ) hydrogel . ( b ) The sarcomeric &alpha - actinin ( green ) , the connexin - 43 ( red ) , and the nuclei ( blue ) on the patterned surface . ( c ) Thr troponin I ( green ) and the nuclei ( blue ) on the unpatterned surface . ( d ) The sarcomeric &alpha - actinin ( green ) , the connexin - 43 ( red ) , and the nuclei ( blue ) on the unpatterned surface . ( e ) The beating behaviour of the cardiomyocytes on the unpatterned surface . ( f ) The beating behaviour of the cardiomyocytes on the patterned surface . ( g ) Spontaneous beating rates of cardiomyocytes on the unpatterned surface . Copyright ( 2014 ) Nature Publishing Group .

The conductive hydrogels ( Chitosan , Polypyrrole ) connect areas to propagate the electrical signal into the entire cardiac tissue . The carbon nanotubes in gelatin methacrylate are actuators in the system ( figure 3 ) . The direction of motion varies as a function of the tubular thickness .

Figure 3 . The actuators were designed by incorporating carbon nanotubes ( CNTs ) in gelatin methacrylate ( GelMA) . ( a ) The tightly and the loosely rolled – up constructs were designed . ( b ) The displacement of the beating constructs are a function of frequency and electrical source . ( c ) The linear displacement of a triangular swimmer as a function of time . Copyright ( 2014 ) Nature Publishing Group .

The cardiac progenitor cells , the mesenchymal stem cells , and the pluripotent stem cells have increased potential for differentiation . The stem cell differentiation direction into the cardiac lineage is indicated by the hydrogel matrix in figure 4 . The immunostaining in the 2D hydrogels demonstrates that the sarcomere has diminished length in comparison to the one in the 3D structure .

Figure 4 . The cardiac patch was designed as cardiomyocytes derived from the human embryonic stem cells ( hESCs ) in the hydrogel matrix . ( a ) The result of 2 weeks of in vitro culture . ( b ) The gels microfacricated with elliptical pores . ( c ) The alignment of the hESCs in the hydrogel . The immunostaining on 3D hydrogel of ( d ) troponin T ( red ) , ( e ) myosin heavy chain ( red ) , ( f ) connexin - 43 ( red ) and sarcomeric α – actinin ( green ) . ( g ) The immunostaining on 2D hydrogel of sarcomeric α – actinin ( green ) . Copyright ( 2014 ) Nature Publishing Group .

The growth factors delivered with cells in the hydrogel may increase the efficiency of the process . Figure 5 presents the effect the basic Fibroblast growth factor ( bFGF ) has in a porcine model . The arteriolar density , the left ventricular ejection fraction , and the myocardial perfusion are increased at 4 weeks in culture , when the bFGF is used .

Figure 5 . The influence of the released rate of basic Fibroblast growth factor ( bFGF ) on restoring the cardiac function in a porcine model of the myocardial infarction . ( a ) The presence of the sarcomeric α – actin , ( b ) the arteriolar density , ( c ) the myocardial perfusion , and ( d ) the left ventricular ejection fraction ( LVEF ) were measured under control and the bFGF included conditions . Copyright ( 2014 ) Nature Publishing Group .

Only 10 % of the cells delivered directly in the cardiac tissue survive in vivo . The direct exposure to oxygen , free radicals , and inflammatory cytokines reduces the survival rate of the injected cells . A suitable 3D microenvironment is required . The hydrogel that mimics the cardiac tissue both handles the cyclic loading forces , and maintains the elastic , in addition to conductive , properties . The oxygen – releasing properties of a porous hydrogel , with or without the use of growth factors , increase the control of the system on the cellular environment .

Cardiomyocytes for myocardial regeneration

The pumping mechanism of the heart is adjusted by the network of coronary arteries and cardiac veins . Myocardial infarction ocurs when the blood supply is disrupted . In the progressive form , this may lead to heart failure .

The repopulation of the scar is achieved through various methods . One of them is to use autologous bone marrow cells . Another strategy is to differentiate the cardiac progenitor cells ( CPC ) . A third direction is to use cardiomyocytes ( CMs ) derived from human embryonic stem cells ( hESC ) , hESCs cardiac progenitor cells ( hESC - CPC ) , human pluripotent stem cells ( hPSCs ) , or human induced pluripotent stem cells ( hiPSCs ) for the transplantation in the myocardium .

The review paper Concise review : reprogramming strategies for cardiovascular regenerative medicine : from induced pluripotent stem cells to direct reprogramming analyses more than a hundred papers . Three reprogramming strategies for the myocardial regeneration are brought in discussion ( figure 1 ) : the iPSCs technology , the partially reprogramming method and the direct set of approaches .

Figure 1 . The CMs derived from the iPSC ( top ) , the partial reprogramming ( middle ) , and the direct reprogramming technology ( below ) .

The hESCs represent the first reliable source for cardiomyocytes in vitro . The immune rejection , and the patient – or disease – specific hESCs are two challenges for the clinical allogeneic cell transplantation .

The hESCs and hiPSCs may generate beating cardiomyocytes in an inefficient method . The canonical Wnt pathway uses hiPSCs and small molecules in a more efficient signaling strategy .

Cardiomyocytes are partially reprogrammed from the iPSCs through a technology developed in 2006 . It takes a few months to differentiate the colonies of hiPSCs into the cardiac lineage . The use of partially reprogrammed cells reduces the length of time for the process to 11 – 12 days . There are also challenges for reaching the clinical practice ( figure 2 ) . Some of them are : the phenotypic heterogeneity of the differentiating CMs , the tumorigenic risk , and the poor in – vivo survival .

Figure 2 . The phases and challenges of the partially reprogrammed iPSCs method towards clinical practice .

The process of direct reprogramming of fibroblasts into induced cardiomyocyte cells ( iCMs ) is epigenetically stable . It is achieved through various combinations : by adding the Hand2 to the Gata4 , Mef2c and Tbx5 combination of transcription factors ; by using myocardin , and a combination of the Mef2c and Tbx5 transcription factors ; through a combination of a Janus inhibitor and four microRNAs ; by mixing two microRNAs and four transcription factors . The challenges of the myocardial regeneration are reduced through the deliver of reprogramming factors directly to the cardiac tissue .

Progress in the regeneration therapy is achieved if further studies are performed on early stages of myocardial infarction , if the stem cells delivered in – vivo generate efficiently new myocardium , and if the newly developed myocardium has suitable performance for the patient .