Stem cells and vascular regenerative medicine: A mini review

2.1 Peripheral arterial disease

Lower limb ischemia causes decreased blood flow in the lower leg with intense pain and swelling. Recently preliminary results of clinical trials using adult stem cell treatment for severe limb ischemia was presented with endothelial progenitor cells (PECs) and mesenchymal stem cells (MSCs). The cells, obtained through bone marrow aspiration, were mixed and infused into damaged vessels. According to this study, there were no adverse effects as a result of the infusions. More importantly, their patients experienced a progressive and lasting improvement in clinical parameters including walking tests oxygen pressure, angiography, and quality of live. Different clinician groops believe that the use of adult stem cell therapy in ischemia patients allows for the development of new mature and stable capillaries. The clinical trials are generally in phase I with autologous mesenchymal stems and mainly in patients with diabetes (Table 1). It can be noted a trials in Scotland with human neural stems in arterial disease.

2.2 Cell therapy and heart disease

Every year in France, around ten thousand new cases of serious cardiac insufficiency are detected. Heart transplants remain the only treatment for the most advanced stages of the condition but the shortage of donors and complications of immunosuppression restrict the indications. Surgical remodelling of the left ventricle only deals with the particular anatomical forms and recent negative results have led to a review of the indications. Mechanical ventricular assistance remains a temporary solution for those waiting for a transplant. There is thus a need for new treatment solutions. Xenotransplantation, despite the hopes raised for it when used with transgenic animals whose organs might be better tolerated by man, is not advancing since the immunological challenges are considerable and there are major safety considerations. Gene therapy is still in its infancy in this area and the complexity of the mechanisms involved in heart failure does not lend itself to this therapeutic approach. Finally, cell therapy no doubt has a place, but only in its intermediate forms, in patients who retain a sufficient reserve of contractile cells. The numerous trials have not made it possible to reach a conclusion at the present time.

Isotypic studies showed the homing of progenitor stem cells from bone marrow towards the lesion sites after a coronary ligation. The molecular signals leading to tissular repair are unknown. It could be some cytokines released during cardiac ischemia.

The treatment of myocardial infarction (MI), however, is subject to a significant constraint: the immediate availability of cells. The intracoronary injection of stem cells prepared starting from a withdrawal of bone marrow did not lead to significant improvements (3% maximum of the ejection fraction of the left ventricle). In the same manner, the intravenous injection of mesenchymal stem cells does not give significant results. In the case of heart failure, the cell therapy turns out to be no efficient and it seems difficult today to envisage a regenerative therapy. At the end of 2007, the U.S. based stem cell company Osiris Therapeutics completed a human trial to use allogeneic stem cells for the treatment for heart disease. An intravenous drip was used to deliver on-self mesenchymal stem cells to patients that had recently suffered a heart attack. No deaths occurred, and the treatment is now widely thought of as safe.

Today it is no regulatory approved cell treatment for myocardial infarction, but research and clinical studies offers the hope for successfull cell therapy in the next decades. It seems that modifications on the fonctional properties of medular cells that arrive in dyslipidemia, diabetes or high blood pressure, wich is found in most of the patients victims of MI. It is possible that the efficacity of the human cardiac therapy in humans depends on the fonctional properties of grafted cells.

2.3 Ischemic stroke

Cerebral infarct is a process, in which brain damage increases with time. Therefore the moment when treatment is started is critical. At present, the only effective treatment (tissular plasminogen activator) has to be administrated very soon after the stroke. In animal models, intravenous administration of huCB cells to rats, after induction of stroke by occlusion of the middle cerebral artery, promoted the improvement of neurological function. The cells were mainly found in the cortex and the striatum of the lesioned hemisphere and outside the brain, in bone marrow, spleen, and in very small amounts in muscle, heart, lungs and liver. These authors found that some of the injected cells showed neuronal markers (NeuN2) and MAP2), astrocytic markers (GFAP), and endothelial cell markers. In another study, huCB were administrated intravenously into the femoral vein or directly into the striatum in order to assess which route of cell administration produced the greatest recovery in rats with permanent middle cerebral artery pluripotency by retroviral transduction of only 2 transcription factors (Oct-4 and Sox-2) without any additional chemical compounds.

Actually, iPS cells should be ideally generated without using viral vectors and without teratoma formation for being suitable for clinical use. The main clinical trials concern autologous bone marrow mesenchymal stem cells.

2.4 Cell therapy and eretctile dysfunctions

Recently, the stem cell therapy for erectile dysfunction has been investigated. Transplantation of stem cells (Adipose-derived stem cells or Bonne Marrow stem cells) was done by intra-cavernous injection. More recent studies used combinatory therapy by supplementing stem cells angiogenic genes or proteins. The different studies reported better erectile function after stem cell mainly by intra-cavernous injection.

The main potential applications are Post-prostatectomy and post-radiotherapy, diabetes associated erectile dysfunction and Lapeyroni’s disease. Human clinical trial of erectile dysfunction with stem cells is not yet approved in most countries. Six trials are now in progress (Table 3).

2.5 Cell therapy and liver diseases

In response to a variety of chronic injuries as hepatitis, alcohol or drug abuse, metabolic diseases, autoimmune attack of hepatocytes or the bile duct epithelium and congenital abnormalities, liver fibrosis occurs and finally results to hepatic cirrhosis and liver failure. Liver transplantation is the well-accepted treatment option for this end-stage liver diseases and acute liver failure resulting in irreversible liver dysfunction. However, it is limited by the shortage of donor organs. Moreover, it’s difficult to accept such a heavy surgical treatment for some patient, the shortage of donor organs. In fact, correction of hepatocyte functional deficiency is the prime goal of liver transplantation in many diseases. There is growing evidence in support of cell therapy. Some authors tried to apply hepatocytes to treat patients with liver diseases instead of liver transplantation. However, the obstacle against their clinical applications is the requirement of large number of hepatocytes that are not available from patients themselves and are not enough from other donors either. Thus, it’s necessary to search for a novel source of cells. Stem cell therapy is now accepted as one of the most promising approach to repopulate liver but the regeneration processes are highly complex. Therefore the idea of using one-type of stem cell to repair liver is acceptable. Various populations of stem cells are under investigation in terms of their regenerative capabilities. Recently, studies showed that extra-hepatic adult mesenchymal stem cells (MSC) of different origins have demonstrated their ability to express a hepatocyte-like phenotype after being differentiated in vitro. These cells include MSC derived from bone marrow, umbilical cord, adipose tissue, placenta are used in 32 trials mainly for cirrhosis (after hepatitis, alcohol abuse and liver transplantation) (Table 4).

3.1 Vascular enginerring

Vascular grafts are today mainly of synthetic or biological origin. Synthetic grafts are most commonly made of Dacron or polytetrafluroethylene. However, their thrombogenic surface and poor mechanical properties limit their use as small-caliber grafts. For this reason, the use of blood vessels (saphenous veins) constitutes an alternative choice for patients in need of small-caliber arterial reconstruction. The use of autografts is nevertheless hampered by limited availability and suitability as the result of extensive peripheral vascular diseases and/or their previous uses in bypass surgery. An alternative vessel source could originate from allograft banks [1–3]. Arterial allografts usually are used for in situ treatment of infected prosthetic grafts [4, 5]. A major advantage related to allografts is their greater abundance compared with autografts and their adequate vascular architecture for small vessel replacements. Nevertheless, cryopreserved arteries exhibit several limits as an increased risk of intimal hyperplasia formation due to a decrease of mechanical properties. Thus, innovative technologies aimed to modify small-diameter natural grafts to obtain functional blood vessels with antithrombogenic and adequate mechanical properties comparable with native vessels constitute challenging research domains and are of the highest clinical importance.

This the layer-by-layer self assembly of polyanions and polycations and the resulting polyelectrolyte multilayer films (PEMs) constitute candidates for tissue engineering applications. This approach also was used for blood vessel coating. Elbert et al. found that the use of polylysine/alginate PEM prevents cell attachment. More recently, Thierry et al. deposited hyaluronan/chitosan films directly on damaged arteries. Moreover, this “in vitro” experiment exhibited very weak platelet and leukocyte adhesion on films ended by a polyanion layer, thus avoiding the thrombogenic process. Menu et al. [71, 115] demonstrated the possibility of depositing a poly(allylamine hydrochloride)/poly(styrene sulfonate) (PAH/PSS) multilayer directly inside arteries that originated from umbilical cords (UCs) and showed that, after the deposition of such a film built with only 3.5 pairs of layers, the initial compliance of the tissue was recovered. Such a recovery is of importance in particular to avoid an intimal hyperplasia at the anastomotic site [114].

3.2 Whole organ engineering

The relevance of research into the creation of reconstructed organs is justified by the lack of organs available for transplant and the growing needs of an ageing population. On a technical level, the development of these reconstructed organs involves two parallel complementary stages: de-cellularization of the target organ with a need to maintain the structural integrity of the extracellular matrix and re-cellularization of the matrix with stem cells or resident cells [8, 95].

Whole organ engineering like liver, kidneys, heart or lung, is particularly difficult because of the structural complexity and heterogeneity of organ cell types. But new ways of researches are currently focused on: the matrix to support re-cellularization and a promising approach is the direct use of extracellular matrix of the organ. Thus rodent, porcine and rhesus monkey organs have been de-cellularized to obtain a scaffold with preserved extracellular matrix and vascular. As concern the source of cells for re–cellularization embryonic, foetal, adult, progenitor stem cells and also IPS have been proposed.

Decellularisation can be achieved through an intra-arterial infusion of a solution containing TritonX-100 and ammonium hydroxide. This method causes all the cellular elements to disappear, leaving elements of the extracellular matrix and the vascular system. Other methods of decellularisation have also been used, employing other chemical, enzymes or physical agents(ultrasound) [51, 159].

Several types of cell can be considered for re-cellularization purposes: stem cells (embryonic and adult stem cells) or patient’s autologous cells. Stem cells probably represent the ideal source of material due to their ability to proliferate. Their use appears limited, nevertheless, by their allogenic nature, which could possibly trigger an immune response and consequent rejection, in addition to the risk with ESC of the formation of teratomes in vivo. These obstacles could be removed in future by using nuclear transfer techniques from the patient’s somatic cells (IPS). Finally, the stem or progenitor cells present in most organs are another source of cells that could be used for in vitro organogenesis. Considered as tools or repair material for their corresponding organs, they often remain difficult to define, isolate and to growth in culture.

Furthermore, the type and number of cells to be used for re-cellularization vary depending on the organ to be reconstructed. Apparently, specific cells of the organ to be reconstructed are indispensable. Other types of cell, such as endothelial cells and fibroblasts are also needed, since they promote the functional cell phenotype and contribute to the structural organisation of tissue. The matrix of the vascular system of the organ to be reconstructed needs to be re-endothelialized so as to orientate the blood flow and prevent thrombosis.

Currently, growing organs in vitro and ex vivo can take several weeks until they have completely developed in the matrix. The seeding methods for re-cellularization are inspired by those used in cell therapy and the most practical solution seems to be intra-arterial infusion. The use of an extra-corporeal pulsating or continuous infusion system (bioreactor) is indispensable for providing the cells with an oxygen supply and keeping the infusate at a constant temperature. The infusion liquids are derived from the culture media used for the cells in question. They need to contain growth factors or other molecules that are more specific to each organ. Finally, there is another hypothetical possibility for re-cellularization, the transplanting of a de-cellularized organ into the recipient, in the hope that re-cellularization will occur directly from the recipient’s owncells.

Encouraging work has recently shown the feasibility of creating bio-organs for the reconstruction of the liver, heart, lungs and kidneys. However, clinical applications still remain a distantprospect.

3.3 Heart

Heart construction could be an alternate option for the treatment of cardiac insufficiency. It is based on the use of an extra-cellular matrix coming from an animal’s heart and seeded with cells likely to reconstruct a normal cardiac function. Though de-cellularization techniques now seem controlled, the issues posed by the selection of the cells capable of generating the various components of cardiac tissue are not settled yet [9, 92]. In addition, the recolonisation of the matrix does not only depend on the phenotype of cells that are used, but it is also impacted by the nature of biochemical signals emitted. The complexity of those problems results in the full replacement of the heart with a biomaterial substitutes to standard transplanting is one prospect. However, it is more realistic to hope, in the medium run, partial replacements of the heart with cellularized matrices reinforcing portions of the failing myocardium or with direct cellular therapy with stem cells [99].

The de-cellularization of animal hearts (rats and, more recently, big mammals) has been performed through the infusion of chemical detergents [129]. The results show that the integrity of the matrix (collagen, fibronectin, laminin, fibre orientation, etc.) can be maintained as well as permeability of the vascular tree and the competence of the heart valves.

Re-cellularization is more problematic due to the diversity of the cell populations that need to be reconstituted. These are the cardiomyocytes, myofibroblasts, endothelial cells and the smooth muscle. Ways to achieve this could be considered [40].

In summary, the complete replacement of a human heart by another heart constituted from a matrix of animal origin and seeded by cells capable to provide the organ with effective, mechanical activity remains a remote prospect and is unlikely to become a reality within the next 10 to 20 years.

An other way is the preparation of cardiac patch. The construction of the high biocompatible biomaterials pre-treated with SC will offer a promising strategy to improve the effects of stem cell therapy for MI [22, 97]. Thus the develop of this cardiac stem cell patch has high therapeutic perspectives for the treatment of MI and prevention of the chronic heart failure. However the materials suitable for the treatment of MI need to have specific quality: biocompatibility, resistant to the mechanical force in situ, suitable for the cell amplification, with suitable size of pores for the cell communication which is necessary for the formation of the functional tissue. Under microscope, the pore size needs to be at least 50μm which is necessary for the vascularization of the patch and assure the MSC metabolism? The biological materials have more advantages than artificial materials because the integration of the cells was optimal for the construction of the cardiac stem cell patch. As the MSC derived from the Wharton Jelly of umbilical cord is easy to be collected, the umbilical artery can be collected preferable at the same time. The natural matrix of the umbilical artery possesses the properties for the construction of a biocompatible cardiac patch.

3.4 Liver

Recent researches have shown that it is possible to use decellularized whole liver treated by detergents as scaffold, which keps the entire network of blood vessels and the integrated extracellular matrix (ECM) [109]. Beside of decellularized whole organ scaffold seeding cells selected to repopulate a decellularized liver scaffold are critical to the function of bioengineered liver. At present, potential cell sources are hepatocyte, mesenchymal stem cells.

Other studies have shown that MSC originated from extre-hepatic tissues can differentiate into endoderm cell-lines as hepatocytes. Several methods have successfully differentiated MSC into hepatocyte, such as stimulating MSC by cytokines an growth factors, direct and indirect co-culture MSC with hepatocytes, or promote MSC differentiation by 3-dimensional matrix. In some cases, differentiation of MSC into hepatocytes can also be an alternative approach for whole organ transplantation in treatment of acute and chronic liver diseases (see Zhang et col. Biomedical materials and engineering 2012; 22:105–111).

Decellularized liver keeps exactly the texture of the orinigal organ. This natural structure can provide a 3-dimensional matrix in favour of cultures cell proliferation, differentiation and function, which promotes the emerge of the idea to use decellularized organ in bioengineering organ (Fig. 1). The liver decellularisation is carried out by perfusing detergents like Triton-X 100 and sodium dodecyl sulphate (SDS), though liver portal vein. This method can destroy cell membrane and take off debris of cells, and at the same time keep the extracellular matric complete with blood and biliary vessels. This matrix maintains the liver-specific proteins proportions fo collagens I and IV, fibronectin and laminin. The intact vasculard system is useful in recellularisation.

3.5 Lungs

About fifty million people throughout the world are living with chronic respiratory failure at a terminal stage. The only treatment for this condition that seriously reduces life expectancy is, in selected cases, lung transplants (first graft 1963) but the results remain poor[98, 101].

A tracheobronchial graft remains a challenge [154]. Research has not yet found an ideal substitute for the airways. Failures have been observed with synthetic prostheses, bio-prostheses, tracheal allografts and autografts. In fact, not only epithelial tissue regeneration but also even cartilaginous regeneration has been observed. Research seems to indicate that this regeneration of tracheal tissue might be possible from an aortic matrix and stem cells taken from bone marrow. Studies have been performed in humans in the context of extended cancer of the trachea and conservation surgery in cases of lung cancer. The research has also contributed prospects for understanding the tissue regenerationmechanisms.

Pulmonary regeneration using stem cells is complex [50, 68]. In fact, several types of local progenitor cells that contribute to cell repair have been described at different levels of the respiratory tract [33, 147]. Moving towards the alveoles, one finds bonchioalveolar stem cells as well as epithelial cells and pneumocytes. In the category of “local stem cells”, cells of the sub-population have been identified that are differentiation markers which in vitro mime stromal mesenchymal cells. The role of these cells in tissue repair has been demonstrated in animal models. Kajstura et al. described resident, multipotent pulmonary stem cells that are capable of self-renewal as well as clonogenicity. The phenotype and functional characteristics of these new cells have been specified in vitro andin vivo.

The lung also contains resident specific mesenchymal stem cells that have recently been described and characterised. These cells do not play a direct part in epithelial renewal but establish communication with the epithelium, thus ensuring their role as a local cytoprotector.

Finally, numerous studies performed on animals have shown the beneficial role played by exogenous, mesenchymal stem cells produced by bone marrow. The effects observed in lesional pulmonary œdema, sepsis, pulmonary hypertension and even idiopathic pulmonary fibrosis have resulted in clinical applications that are currently being assessed. The immuno-modulatory, anti-inflammatory, anti-apoptotic and angiogenic properties of MSC today place these cells at the heart of tissue repair. Contrary to past hypotheses, these cells do not seem to differentiate themselves into alveolar epithelial cells and their method of action would involve paracrine mechanisms not all of which have as yet beenexplained.

These initial researches open up a promising route for developing a functional bio-artificial lung, with the prospect of application to humans within 15 to 20 years. However many questions remain to be answered: Is the use of a decellularized pulmonary matrix the only possible solution? Which cells should be chosen for recellularisation?: (Mesenchymatous stem cells? Resident pulmonary cells?) What is the optimal length of time for incubation in a bioreactor? Would the technique be applicable to the human lung with its very extensive alveolar surface?

3.6 Kidney

The kidney is certainly one of the most difficult organs to reconstruct due to its complex nature and the heterogeneous nature of the cells from which it is constituted [111, 133]. There is relatively little research on auto-construction, though experiments have been performed on rats, pigs and monkeys [15, 112]. The first demonstration of the feasibility of the technique was realised in the rat. His team introduced extracellular matrices using embryonic stem cells injected into the renal artery or the ureter. The cells introduced were differentiated into glomerular, tubular and vascular structures. They nevertheless lost their embryonic phenotype as can be seen from the appearance of immuno-histochemical markers. Nakayama decellularised sections of the kidneys taken from macaques at various growth stages from foetus to adult, via intermediate ages, with the aim of optimising decellularisation techniques and recellularisation in vitro. He demonstrated that the appearance of Pax-2 and Vimentim markers after the cells had been implanted originated from the kidneys of thefoetus.

As with all organs, research into the construction of a kidney raises numerous questions about the preparation of a matrix and the sources of the cells destined for recellularisation. Biological matrices have proved their superiority over the synthetic matrices sometimes used in tissue engineering. In the case of the kidney, the most frequently employed matrices are allogenic, even though xenogenic matrices could be considered, although they might be subject to specific immunological and regulatory issues.

A large number of problems remain to be resolved before a kidney can be prepared or constructed from an extra-cellular matrix. Furthermore, none of the ‘self-constructed’ organs in animals have proved capable of performing the vital function in the recipient for longer than a few hours. In the case of the kidney, no transplant has yet been reported even though it is the main challenge for research. The objective remains plausible, however, even if clinical applications appear to be very remote, certainly not before 15 to 20 years.