Organ reconstruction: Dream or reality for the future.

The relevance of research on reconstructed organs is justified by the lack of organs available for transplant and the growing needs for the ageing population. The development of a reconstructed organ involves two parallel complementary steps: de-cellularization of the organ with the need to maintain the structural integrity of the extracellular matrix and vascular network and re-cellularization of the scaffold with stem cells or resident cells.Whole organ engineering for liver, heart, lung or kidneys, is particularly difficult because of the structural complexity of organs and heterogeneity of cells. Rodent, porcine and rhesus monkey organs have been de-cellularized to obtain a scaffold with preserved extracellular matrix and vascular network. As concern the cells for re-cellularization, embryonic, foetal, adult, progenitor stem cells and also iPS have been proposed.Heart construction could be an alternative 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 cells capable of generating the various components of cardiac tissue are not settled yet. 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.Recent researches have shown that it is possible to use decellularized whole liver treated by detergents as scaffold, which keeps the entire network of blood vessels and the integrated extracellular matrix (ECM). Beside of decellularized whole organ scaffold seeding cells selected to repopulate a decellularized liver scaffold are critical for the function of the bioengineered liver. At present, potential cell sources are hepatocyte, and mesenchymal stem cells.Pulmonary regeneration using engineering approaches is complex. In fact, several types of local progenitor cells that contribute to cell repair have been described at different levels of the respiratory tract. Moving towards the alveoles, one finds bronchioalveolar stem cells as well as epithelial cells and pneumocytes. A promising option to increase the donor organ pool is to use allogeneic or xenogeneic decellularized lungs as a scaffold to engineer functional lung tissue ex vivo.The kidney is certainly one of the most difficult organs to reconstruct due to its complex nature and the heterogeneous nature of the cells. There is relatively little research on auto-construction, and experiments have been performed on rats, pigs and monkeys.Nevertheless, before these therapeutic approaches can be applied in clinical practice, many researches are necessary to understand and in particular the behaviour of cells on the decellularized organs as well as the mechanisms of their interaction with the microenvironment. Current knowledges allow optimism for the future but definitive answers can only be given after long term animal studies and controlled clinical studies.

Stem cells and vascular regenerative medicine: A mini review.

Most human tissues do not regenerate spontaneously, which is why "cell therapy" are promising alternative treatments. The Principe is simple: patients' or donors' cells are collected and introduced into the injured tissues or organs directly or in a porous 3D material, with or without modification of their properties. This concept of regenerative medicine is an emerging field which can be defined as "the way to improve health and quality of life by restoring, maintaining, or enhancing tissue and organ functions".There is an extraordinarily wide range of opportunities for clinical applications: artheropathies, diabetes, cartilage defects, bone repair, burns, livers or bladder regeneration, organs reconstruction (lung, heart, liver ...) neurodegenerative disorders, sepsis ...  Different stem cells (SC) with different potential can be used and characterised (totipotent, mesenchymal of different origins, especially those present in tissues...). Today it is undeniable that cells like bone marrow, adipose tissue or Wharton Jelly stem cells, are of potential interest for clinical applications because they are easily separated and prepared and no ethical problems are involved in their use.In this paper some potential clinical applications in the vascular field are considered: peripheral arteriopathy in diabetic patients, cardiac insufficiency, traitment of erectile dysfunction, or organ regeneration with liver as example. But the regeneration of tissue or organ is and will remain a challenge for the future development of cell therapy. Many problems remain to be solved that could lead to the development of innovative strategies to facilitate cell differentiation, increase the yield of cells and ensure a standardised product, overcome the risks of teratogenic effects and/or immune reactions, enable grafting via direct cell or biotissue transplantation and avoid legal issues involved in national regulations.

Stem Cells and Regenerative Medicine: Myth or Reality of the 21th Century.

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been an increasing interest in the study of undifferentiated progenitors that have the ability to proliferate and differentiate into various tissues. Stem cells (SC) with different potency can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult or even fetal stem cells provide a more interesting approach for clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue, or Wharton's Jelly are of potential interest for clinical applications in regenerative medicine because they are easily available without ethical problems for their uses. During the last 10 years, these multipotent cells have generated considerable interest and have particularly been shown to escape to allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone and cartilage deterioration, diabetes, urology, liver, ophthalmology, and organ's reconstruction). This review focuses mainly on tissue and organ regeneration using SC and in particular MSC.

Signalling pathways involved in the process of mesenchymal stem cells differentiating into hepatocytes.

End-stage liver disease can be the termination of acute or chronic liver diseases, with manifestations of liver failure; transplantation is currently an effective treatment for these. However, transplantation is severely limited due to the serious lack of donors, expense, graft rejection and requirement of long-term immunosuppression. Mesenchymal stem cells (MSCs) have attracted considerable attention as therapeutic tools as they can be obtained with relative ease and expanded in culture, along with features of self-renewal and multidirectional differentiation. Many scientific groups have sought to use MSCs differentiating into functional hepatocytes to be used in cell transplantation with liver tissue engineering to repair diseased organs. In most of the literature, hepatocyte differentiation refers to use of various additional growth factors and cytokines, such as hepatocyte growth factor (HGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), oncostatin M (OSM) and more, and most are involved in signalling pathway regulation and cell-cell/cell-matrix interactions. Signalling pathways have been shown to play critical roles in embryonic development, tumourigenesis, tumour progression, apoptosis and cell-fate determination. However, mechanisms of MSCs differentiating into hepatocytes, particularly signalling pathways involved, have not as yet been completely illustrated. In this review, we have focused on progress of signalling pathways associated with mesenchymal stem cells differentiating into hepatocytes along with the stepwise differentiation procedure.

Stem cells and applications: a survey.

Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been increasing interest in the study of undifferentiated progenitors that have ability to proliferate and differentiate in different tissues. Different stem cells (SC) with different potential can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult stem cells provide a more interesting approach to clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue or MSC of Wharton Jelly, which have limited potential, are of interest for clinical applications in regenerative medicine because they are easily separated and prepared and no ethical problems are involved in their use.During the last 10 years, these multipotent cells have generated considerable interest and in particular have been shown to escape allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone, cartilage, diabetes, ophthalmology, urology, liver, organ's reconstruction…).

Hydroxyapatite incorporated into collagen gels for mesenchymal stem cell culture.

Collagen gels could be used as carriers in tissue engineering to improve cell retention and distribution in the defect. In other respect hydroxyapatite could be added to gels to improve mechanical properties and regulate gel contraction. The aim of this work was to analyze the feasibility to incorporate hydroxyapatite into collagen gels and culture mesenchymal stem cells inside it. Human bone marrow mesenchymal stem cells (hMSC-BM) were used in this study. Gels were prepared by mixing rat tail type I collagen, hydroxyapatite microparticles and MSCs. After polymerization gels were kept in culture while gel contraction and mechanical properties were studied. In parallel, cell viability and morphology were analyzed. Gels became free-floating gels contracted from day 3, only in the presence of cells. A linear rapid contraction phase was observed until day 7, then a very slow contraction phase took place. The incorporation of hydroxyapatite improved gel stability and mechanical properties. Cells were randomly distributed on the gel and a few dead cells were observed all over the experiment. This study shows the feasibility and biocompatibility of hydroxyapatite supplemented collagen gels for the culture of mesenchymal stem cells that could be used as scaffolds for cell delivery in osteoarticular regenerative medicine.

Human stem cells and articular cartilage tissue engineering.

Injuries to articular cartilage are one of the most challenging issues of musculoskeletal medicine due to the poor intrinsic ability of this tissue for repair. Despite progress in orthopaedic surgery, cell-based surgical therapies such as autologous chondrocyte transplantation (ACT) have been in clinical use for cartilage repair for over a decade but this approach has shown mixed results. Moreover, the lack of efficient modalities of treatment for large chondral defects has prompted research on cartilage tissue engineering combining cells, scaffold materials and environmental factors. This paper focuses on the main parameters in tissue engineering and in particular, on the potential of mesenchymal stem cells (MSCs) as an alternative to cells derived from patient tissues in autologous transplantation and tissue engineering. We discussed the prospects of using autologous chondrocytes or MSCs in regenerative medicine and summarized the advantages and disadvantages of these cells in articular cartilage engineering.

French and European regulations for tissue and cell therapy.

This article is focused on the current European and French regulations from a tissue and cell therapy perspective. The first part covers the different Directives of the European Parliament such as the 2004/23/CE and the 2006/17/CE that are applied in France through different Laws (2011-814 Bioethics), Decrees and Orders. The French 2007-1220 Decree sets a framework for science-oriented research as opposed to the 2008-968 Decree that applies to therapy-oriented organizations. The French good manufacturing practices that apply to tissue and cells were published in October 2010, they have been applicable for all tissue and cellular therapy product processing facilities. The sole purpose of all these regulations is to promote good clinical care by increasing safety and control at every single stage of the tissue and cell therapy lifecycle.

Introduction to regenerative medicine and tissue engineering.

Human tissues don't regenerate spontaneously, explaining why regenerative medicine and cell therapy represent a promising alternative treatment (autologous cells or stem cells of different origins). The principle is simple: cells are collected, expanded and introduced with or without modification into injured tissues or organs. Among middle-term therapeutic applications, cartilage defects, bone repair, cardiac insufficiency, burns, liver or bladder, neurodegenerative disorders could be considered.

SHG as a new modality for large field of view imaging to monitor tissue collagen network.

For this study, we have considered a new large field of view imaging dedicated to matrix collagen (no stained samples). To integrate a multidimensional scale (non-sliced samples), a femtosecond oscillator (two photon excitation laser) has been coupled with a large field optical setup to collect SHG signal. We introduced an index (F-SHG) based on decay time response measured by TCSPC for, respectively, Fluorescence (F) and Second Harmonic Generation (SHG) values. For samples where protein collagen is the major component of extracellular matrix (skin) or not (nacre), we compared the index distribution (from 2 to 12) obtained with large field optical setup. In this work, we showed for the first time that multiscale large field imaging combined to multimodality approaches (SHG-TCSPC) could be an innovative and non invasive technique to detect and identify some biological interest molecules (collagen) in biomedical topics.

Introduction to tissue engineering and application for cartilage engineering.

Tissue engineering is a multidisciplinary field that applies the principles of engineering, life sciences, cell and molecular biology toward the development of biological substitutes that restore, maintain, and improve tissue function. In Western Countries, tissues or cells management for clinical uses is a medical activity governed by different laws. Three general components are involved in tissue engineering: (1) reparative cells that can form a functional matrix; (2) an appropriate scaffold for transplantation and support; and (3) bioreactive molecules, such as cytokines and growth factors that will support and choreograph formation of the desired tissue. These three components may be used individually or in combination to regenerate organs or tissues. Thus the growing development of tissue engineering needs to solve four main problems: cells, engineering development, grafting and safety studies.

Polyelectrolyte multilayer films promote human cord blood stem cells differentiation into mature endothelial cells exhibiting a stable phenotype.

BACKGROUND: recent studies in bio-engineering have showed the influence of Polyelectrolyte Multilayer (PEM) films on endothelial cells (ECs), especially poly(sodium-4-styrene-sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). They were tested either with human mature ECs or rabbit immature endothelial progenitor cells (EPCs), but never on human EPCs. In view to obtain an EC covered surface, human cord blood (HCB) EPCs were cultivated on PSS/PAH films. MATERIAL AND METHODS: PEMs were obtained by 7 alternate depositions of cationic PAH and anionic PSS layers. HCB mononuclear cells were isolated by centrifugation through density gradient. 7 days after seeding on PEM, unattached cells were removed and adherent EPCs were cultivated in endothelial specific medium until P6. Appearance of CD31 and vWF was evaluated by confocal microscopy. RESULTS: EPCs not only successfully adhered on PEM, but also spread and proliferated. Moreover, cells differentiated into a typical endothelial cobblestone monolayer within 2 weeks. Immunostaining of CD31 and vWF confirmed the formation of an EC-like confluent monolayer. Furthermore, these cells showed after 6 passages a good phenotypic stability while reseeded on the PEM film. CONCLUSION: these results show an easy way to obtain mature ECs from human stem cells, which may open new applications for a scaffold cellularization in tissue bio-engineering.

Quantification of residual dimethylsulfoxide after washing cryopreserved stem cells and thawing tissue grafts.

Dimethylsulfoxide (DMSO) is a cryoprotective substance often used to allow long term storage of stem cells or tissue grafts. However, a high frequency of adverse events is associated with the infusion of thawed cells. These events are in part due to DMSO, leading many cell therapy facilities to introduce a washing step before the delivery of the grafts. The lack of method for evaluating the residual quantities of this substance in the reinfused cells led us to develop a technique, based on capillary zone electrophoresis for assaying DMSO. The cryoprotectant was measured in 55 hematopoietic stem cell grafts, 6 parathyroids and 5 blood vessels immediately after thawing and after washing or bathing in a saline solution. The results showed that DMSO reduction in stem cell grafts reached more than 90% after the washing procedure. Furthermore, this study has shown that 2 washing steps significantly improved DMSO elimination as compared to 1 washing step. For parathyroids and blood vessels, bathing the tissues after thawing in a saline solution allowed more than 95% DMSO reduction. This study demonstrated that the technique of DMSO measurement used here, is simple and feasible on complex matrices such as protein samples after dilution. It is an appropriate method for residual quantification of the cryoprotectant before graft.

Three-dimensional sprayed active biological gels and cells for tissue engineering application.

Complex three-dimensional structures can "a priori" be built layer-by-layer with a large number of different components, including various cell types, polyelectrolytes, drugs, proteins, peptides or DNA. Our approach is based on the spraying of such elements in order to form a highly functionalized and structured biomaterial. The proposed route will allow the control at the surface and in depth the distribution of the different included elements (matrix and cells).The main objective of this work concerns the buildup of biomaterials aimed to reconstruct biological tissue. The proposed ways are highly innovative and consist in a simple and progressive spraying of all the elements constituting finally the biomaterial.We report here that it is possible (i) to build an alginate gel by alternate spraying of alginate and Ca(2+); (ii) to spray active alginate gel and cells; (iii) to build layer-by-layer an active reservoir under and on the top of this sprayed gel and cells; (iv) to follow the activity of these sprayed cells with time; (v) to propose a three-dimensional sprayed structure for tissue engineering application.

Review: behaviour of endothelial cells faced with hypoxia.

Hypoxia is a diminution of oxygen quantity delivered to tissue for cellular need to product energy. Hypoxia derives from two major conditions in health diseases: anemia and ischemia. Anemic hypoxia comes from damage to O(2) transport like red blood cells diminution or disease. Ischemic hypoxia is a diminution of blood flow following a diminution of blood volume after a hemorrhagic shock. After hypoxia, vessels dilate to increase blood flow allowing a better oxygenation of peripheral tissues. This vasodilation appears immediately after the beginning of hypoxia and can be maintained during several hours. Today, the molecular mechanisms of this vasodilation stay unclear. But it seems that potassic channels, ATP concentration and medium acidification in addition to vasodilator/vasoconstrictor balance play a great role to facilitate the oxygenation of the ischemic areas.As endothelial cells (EC) are lining the vasculature, they are always in contact with blood, which carries, amongst other compounds, oxygen. In this way, they are the first target for an oxygen partial pressure (PO(2)) diminution. EC, through different mechanosensors, can sense a variation in PO(2) and adapt their metabolism to maintain ATP production. Under hypoxia, EC switch into hypoxic metabolism, leading to the production of reactive oxygen species (ROS). Indeed, when PO(2) is low, the respiratory chain in the mitochondria runs slower. Furthermore, cytochrome C capacity to trap O(2) is reduced; this phenomenon alters the cellular redox potential and leads to the accumulation of electrons that induce the formation of ROS.This review presents an overview of the behaviour of endothelial cells face to hypoxia. We propose to focus on nitric oxide, hypoxia inducible factor (HIF), lactate and ROS productions. Then we present the different mode of culture of EC under hypoxia. Finally, we conclude on the difficulty to study hypoxia because of the various types of system developed to reproduce this phenomenon and the different signalling ways that can be activated.

Chimerism analysis following nonmyeloablative stem cell transplantation using a new cell subset separation method: Robosep.

Chimerism analysis has become an important tool to manage patients in the peri-transplant period of allogenic stem cell transplantation. During this period, cells of donor and host origin can coexist and increasing proportion of cells of host origin is considered as a recurrence of the underlying disease. We currently performed chimerism analysis on separate peripheral blood cell subsets, lymphocytes and granulocytes. To improve our isolation method, a new automated device from Stem Cell Technology Roboseptrade mark was tested and compared to our manual separation technique. The results obtained on T cell purification showed an improvement of the purity (98.42% with Robosep vs. 92.42% with the manual technique Rosettesep) and of the recovery (63.43% with Robosep and 38% with Rosettesep). The results were significantly improved on patient samples with less than 10% CD3 positive cells (purity: 90% vs. 44.44%; recovery: 73.79% vs. 43.98%). Granulocytes separation was based on CD15 expression. The results showed an improvement of the purity with Robosep (96.90% vs. 86.20% with the manual technique Polymorphprep) but the recovery was impaired (35.2% vs. 52.30%). Using a myeloid (CD66/CD33) cocktail, recovery was improved with the Robosep device (64.04% with the myeloid cocktail vs. 22.4% with the CD15 cocktail). Our data demonstrated that Robosep allowed a performant cell purification in the early period post-transplantation even for populations representing less than 10% of the peripheral blood cells.

In vitro impact of physiological shear stress on endothelial cells gene expression profile.

In the vascular system, the shear applied to the vascular wall activates mechano-sensors located on endothelial cells (ECs) leading to a modification in the gene expression profile. We applied laminar shear stress at 1 Pa on ECs for 6 h and measured by quantitative real time PCR the expression modulation of genes implied in inflammation (ICAM-1 and E-selectin), oxidative stress sensing (HO-1) and vascular tone modulation (eNOS). We showed that all these genes are shear stress inducible. ICAM-1 is more up-regulated than E-selectin suggesting different levels of implication in inflammatory responses and different modes of induction (SSRE, cytokine). Laminar shear stress induces an oxidative stress translated into HO-1 up-regulation, and a possible vasodilatation through the induction of eNOS. Our laminar shear stress system opens a novel and interesting frame in the evaluation of the impact on ECs and blood cells of new pharmacological substances injected in the bloodstream.

Human umbilical vein endothelial cells increase ex vivo expansion of human CD34(+) PBPC through IL-6 secretion.

BACKGROUND: Ex vivo expansion of hematopoietic stem cells (HSC) can help reduce cytopenia following transplantation, especially in NHL patients whose BM is deficient because of extensive chemotherapy. We have previously reported that human umbilical vein endothelial cells (HUVEC) can contribute to improved PBPC expansion when used in co-culture with CD34(+) cells. METHODS: We evaluated the roles of direct HUVEC CD34(+) contact and HUVEC-produced soluble factors. We cultured CD34(+) PBPC harvested from NHL patients in four different conditions: (1) liquid culture without HUVEC; (2) co-culture in contact with HUVEC; (3) co-culture with HUVEC but without direct contact; (4) liquid culture with HUVEC-conditioned medium (CM). Thrombopoietin (Tpo), Flk2Flt3 ligand (FL) and c-kit ligand (KL) with or without rhIL-6 were added to these four culture conditions. RESULTS AND DISCUSSION: Our results showed that HUVEC co-culture or addition of HUVEC-CM to Tpo, FL and KL (TFK) improved CD34(+) PBPC expansion compared with liquid culture, as determined by total viable nucleated cells (TNC), colony-forming cell assay (CFC) and week-6 cobblestone area-forming cells (Wk-6 CAFC) expansions. Non-contact culture led to similar PBPC expansion as contact co-culture; moreover, HUVEC-CM improved PBPC expansion. However, when rhIL-6 was added to HUVEC-CM with TFK, no significant difference was observed. Finally, high quantities of IL-6 were detected in HUVEC-CM and addition of anti-IL-6 Ab inhibited the positive effect of HUVEC on PBPC expansion. Our results thus suggest that HUVEC may improve PBPC expansion, at least through IL-6 secretion.