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Hematopoietic stem and progenitor cells (HSPCs) give rise to all cell types of the hematopoietic system through various processes, including asymmetric divisions. However, the contribution of stromal cells of the hematopoietic niches in the control of HSPC asymmetric divisions remains unknown. Using polyacrylamide microwells as minimalist niches, we show that specific heterotypic interactions with osteoblast and endothelial cells promote asymmetric divisions of human HSPCs. Upon interaction, HSPCs polarize in interphase with the centrosome, the Golgi apparatus, and lysosomes positioned close to the site of contact. Subsequently, during mitosis, HSPCs orient their spindle perpendicular to the plane of contact. This division mode gives rise to siblings with unequal amounts of lysosomes and of the differentiation marker CD34. Such asymmetric inheritance generates heterogeneity in the progeny, which is likely to contribute to the plasticity of the early steps of hematopoiesis.
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Células Madre Hematopoyéticas , Humanos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Hematopoyesis/fisiología , Diferenciación Celular , Mitosis , Osteoblastos/citología , Osteoblastos/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , División Celular Asimétrica , Lisosomas/metabolismo , Centrosoma/metabolismo , Antígenos CD34/metabolismo , Aparato de Golgi/metabolismo , División CelularRESUMEN
Hematopoietic stem and progenitor cells (HSPC) can differentiate into all hematopoietic lineages to support hematopoiesis. Cells from the myeloid and lymphoid lineages fulfill distinct functions with specific shapes and intra-cellular architectures. The role of cytokines in the regulation of HSPC differentiation has been intensively studied but our understanding of the potential contribution of inner cell architecture is relatively poor. Here, we show that large invaginations are generated by microtubule constraints on the swelling nucleus of human HSPC during early commitment toward the myeloid lineage. These invaginations are associated with a local reduction of lamin B density, local loss of heterochromatin H3K9me3 and H3K27me3 marks, and changes in expression of specific hematopoietic genes. This establishes the role of microtubules in defining the unique lobulated nuclear shape observed in myeloid progenitor cells and suggests that this shape is important to establish the gene expression profile specific to this hematopoietic lineage. It opens new perspectives on the implications of microtubule-generated forces, in the early commitment to the myeloid lineage.
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Diferenciación Celular , Expresión Génica , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Microtúbulos , Línea Celular , Linaje de la Célula , Núcleo Celular/genética , Núcleo Celular/fisiología , Citocinas , Células Madre Hematopoyéticas/citología , Histonas , Humanos , TranscriptomaRESUMEN
Sustained ANKRD26 expression associated with germline ANKRD26 mutations causes thrombocytopenia 2 (THC2), an inherited platelet disorder associated with a predisposition to leukemia. Some patients also present with erythrocytosis and/or leukocytosis. Using multiple human-relevant in vitro models (cell lines, primary patients' cells and patient-derived induced pluripotent stem cells) we demonstrate for the first time that ANKRD26 is expressed during the early steps of erythroid, megakaryocyte and granulocyte differentiation, and is necessary for progenitor cell proliferation. As differentiation progresses, ANKRD26 expression is progressively silenced, to complete the cellular maturation of the three myeloid lineages. In primary cells, abnormal ANKRD26 expression in committed progenitors directly affects the proliferation/differentiation balance for the three cell types. We show that ANKRD26 interacts with and crucially modulates the activity of MPL, EPOR and G-CSFR, three homodimeric type I cytokine receptors that regulate blood cell production. Higher than normal levels of ANKRD26 prevent the receptor internalization that leads to increased signaling and cytokine hypersensitivity. These findings afford evidence how ANKRD26 overexpression or the absence of its silencing during differentiation is responsible for myeloid blood cell abnormalities in patients with THC2.
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Leucemia , Receptores de Citocinas , Humanos , Citocinas , Hematopoyesis , Leucemia/patología , Diferenciación Celular , Péptidos y Proteínas de Señalización IntercelularRESUMEN
In pathologies of the esophagus such as esophageal atresia, cancers and caustic injuries, methods for full thickness esophageal replacement require the sacrifice of healthy intra-abdominal organs such as the stomach and the colon. These methods are associated with high morbidity, mortality and poor functional results. The reconstruction of an esophageal segment by tissue engineering (TE) could answer this problem. For esophageal TE, this approach has been explored mainly by a combination of matrices and cells. In this chapter, we will discuss the studies on full organ esophageal decellularization, including the animal models, the methods of decellularization and recellularization.
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Ingeniería de Tejidos , Andamios del Tejido , Animales , Colon , EsófagoRESUMEN
After 30 years of hematopoietic stem cell use for various indications, umbilical cord blood is considered as an established source of cells with marrow and postmobilization peripheral blood. The limited number of cells still remains a problematic element restricting their use, especially in adults who require to be grafted with a higher cell number. Improving the quality of harvested cord blood, at least in terms of volume and amount of cells, is essential to decrease the number of discarded units. In this review, we examine several variables related to parturient, pregnancy, labor, delivery, collection, the newborn, umbilical cord, and placenta. We aim to understand the biologic mechanisms that can impact cord blood quality. This knowledge will ultimately allow targeting donors, which could provide a rich graft and improve the efficiency of the collection.
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Conservación de la Sangre , Trasplante de Células Madre de Sangre del Cordón Umbilical , Sangre Fetal , Aloinjertos , HumanosRESUMEN
Tissue engineering holds great promise for regenerative medicine, drug discovery, and as an alternative to animal models. However, as soon as the dimensions of engineered tissue exceed the diffusion limit of oxygen and nutriments, a necrotic core forms leading to irreversible damage. To overcome this constraint, the establishment of a functional perfusion network is essential. In this work, digital light processing bioprinting is used to encapsulate endothelial progenitor cells (EPCs) in 3D light-cured hydrogel scaffolds to guide them toward vascular network formation. In these scaffolds, EPCs proliferate and self-organize within a few days into branched tubular structures with predefined geometry, forming capillary-like vascular tubes or trees of diameters in the range of 10 to 100 µm. Presenting a confluent monolayer wall of cells strongly connect by tight junctions around a central lumen-like space, these structures can be microinjected with a fluorescent dye and are stable for several weeks in vitro. These endothelial structures can be recovered and manipulated in an alginate patch without altering their shape or viability. This approach opens new opportunities for future applications, such as stacking with other cell sheets or multicellular constructs to yield bioengineered tissue with higher complexity and functionality.
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Bioimpresión , Células Progenitoras Endoteliales , Ingeniería de Tejidos , Andamios del Tejido , Humanos , Bioimpresión/métodos , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Células Progenitoras Endoteliales/citología , Células Progenitoras Endoteliales/metabolismo , Hidrogeles/química , Capilares/fisiología , Alginatos/química , Impresión TridimensionalRESUMEN
Changing practices and the limited use of cord blood units as a source of cells for allogeneic hematopoietic stem cell transplants (HSC) led us to reconsider the recommendations established in 2011 and 2012, and to propose an update incorporating recent bibliographic data. If HLA compatibility was until now established at low resolution for HLA-A and B loci, and at high resolution for HLA-DRB1, the recent papers are converging towards an increase in the level of resolution, making way for a compatibility now defined in high resolution for all the considered loci, and the inclusion of the HLA-C locus, in order to establish a level of HLA compatibility on 8 alleles (HLA-A, B, C and DRB1). The CD34+ dose is a determining factor in hematopoietic reconstitution but it is not correlated with the total nucleated cells content. This is why we recommend taking these two data into account when choosing a cord blood unit. The recommendations established by our group are presented as a flow chart taking into account the characteristics of the underlying pathology (malignant or non-malignant), the cell dose and the HLA compatibility criteria, as well as criteria linked to the banks in which units are stored.
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Tissue engineering is a promising alternative to current full thickness circumferential esophageal replacement methods. The aim of our study was to develop a clinical grade Decellularized Human Esophagus (DHE) for future clinical applications. After decontamination, human esophagi from deceased donors were placed in a bioreactor and decellularized with sodium dodecyl sulfate (SDS) and ethylendiaminetetraacetic acid (EDTA) for 3 days. The esophagi were then rinsed in sterile water and SDS was eliminated by filtration on an activated charcoal cartridge for 3 days. DNA was removed by a 3-hour incubation with DNase. A cryopreservation protocol was evaluated at the end of the process to create a DHE cryobank. The decellularization was efficient as no cells and nuclei were observed in the DHE. Sterility of the esophagi was obtained at the end of the process. The general structure of the DHE was preserved according to immunohistochemical and scanning electron microscopy images. SDS was efficiently removed, confirmed by a colorimetric dosage, lack of cytotoxicity on Balb/3T3 cells and mesenchymal stromal cell long term culture. Furthermore, DHE did not induce lymphocyte proliferation in-vitro. The cryopreservation protocol was safe and did not affect the tissue, preserving the biomechanical properties of the DHE. Our decellularization protocol allowed to develop the first clinical grade human decellularized and cryopreserved esophagus.
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Matriz Extracelular , Andamios del Tejido , Ratones , Animales , Humanos , Andamios del Tejido/química , Ingeniería de Tejidos/métodos , Criopreservación , Dodecil Sulfato de Sodio/química , EsófagoRESUMEN
BACKGROUND: Myelomeningocele (MMC) is a spinal cord congenital defect that leads to paraplegia, sphincter disorders and potential neurocognitive disabilities. Prenatal surgery of MMC provides a significant benefit compared to surgery at birth. Mesenchymal stromal cell (MSC) therapy as an adjuvant treatment for prenatal surgery showed promising results in animal experiments which could be considered for clinical use in human fetuses. Despite numerous reassuring studies on the safety of MSCs administration in humans, no study focused on MSCs biodistribution after a local MSCs graft on the fetal spinal cord. AIM: The purpose of our study was to assess the biodistribution of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) at birth in lambs who had a prenatal myelomeningocele repair using a fibrin patch seeded with allogenic UC-MSCs. METHODS: After isolation, UC-MSCs were tagged using a green fluorescent protein (GFP)-containing lentiviral vector. MMC defects were surgically created at 75 days of gestation and repaired 15 days later using UC-MSCs patch. Lambs were delivered at 142 days and sacrificed. DNA extraction was performed among biopsies of the different organs and q-PCR analysis was used to detect the expression of GFP (GFP DNA coding sequence). RESULTS: In our 6 surviving lambs grafted with UC-MSCs, GFP lentivirus genomic DNA was not detected in the organs. CONCLUSION: These reassuring data will support translational application in humans, especially since the first human clinical trial using mesenchymal stromal cells for in-utero treatment of MMC started recently in U.S.A.
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Meningomielocele , Células Madre Mesenquimatosas , Animales , Femenino , Feto/metabolismo , Humanos , Meningomielocele/metabolismo , Meningomielocele/cirugía , Células Madre Mesenquimatosas/metabolismo , Embarazo , Ovinos , Oveja Doméstica , Distribución Tisular , Cordón Umbilical/metabolismoRESUMEN
BACKGROUND: Tissue engineering is an attractive alternative to conventional esophageal replacement techniques using intra-abdominal organs which are associated with a substantial morbidity. The objective was to evaluate the feasibility of esophageal replacement by an allogenic decellularized esophagus in a porcine model. Secondary objectives were to evaluate the benefit of decellularized esophagus recellularization with autologous bone marrow mesenchymal stromal cells and omental maturation of the decellularized esophagus. METHODS: Eighteen pigs divided into 4 experimental groups according to mesenchymal stromal cells recellularization and omental maturation underwent a 5-cm long circumferential replacement of the thoracic esophagus. Turbo green florescent protein labelling was used for in vivo mesenchymal stromal cells tracking. The graft area was covered by a stent for 3 months. Clinical and histologic outcomes were analyzed over a 6-month period. RESULTS: The median follow-up was 112 days [5; 205]. Two animals died during the first postoperative month, 2 experienced an anastomotic leakage, 13 experienced a graft area stenosis following stent migration of which 3 were sacrificed as initially planned after successful endoscopic treatment. The stent could be removed in 2 animals: the graft area showed a continuous mucosa without stenosis. After 3 months, the graft area showed a tissue specific regeneration with a mature epithelium and muscular cells. Clinical and histologic results were similar across experimental groups. CONCLUSION: Circumferential esophageal replacement by a decellularized esophagus was feasible and allowed tissue remodeling toward an esophageal phenotype. We could not demonstrate any benefit provided by the omental maturation of the decellularized esophagus nor its recellularization with mesenchymal stromal cells.
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Esófago/anatomía & histología , Esófago/cirugía , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Estudios de Factibilidad , Femenino , Trasplante de Células Madre Mesenquimatosas , Modelos Animales , Epiplón/citología , Stents , Porcinos , Trasplante AutólogoRESUMEN
PURPOSE OF THE STUDY: The purpose of our study was to investigate the effects of ovine umbilical cord-derived mesenchymal stromal cells (UC-MSCs) seeded in a fibrin patch as an adjuvant therapy for fetal myelomeningocele repair in the ovine model. MATERIALS AND METHODS: MMC defects were surgically created at 75 days of gestation and repaired 15 days later with UC-MSCs patch or an acellular patch. At birth, motor function, tail movements, and voiding abilities were recorded. Histological and immunohistochemical analysis included study of MMC defect's healing, spinal cord, UC-MSCs survival, and screening for tumors. RESULTS: Six lambs were born alive in each group. There was no difference between the two groups on the median sheep locomotor rating score but all lambs in the control group had a score between lower than 3 compared to 50% in UC-MSCs group. There were more lambs with tail movements and voiding ability in UC-MSCs group (83% vs 0% and 50% vs 0%, respectively). gray matter area and large neurons density were higher in UC-MSCs group (2.5 vs 0.8 mm2 and 19.3 vs 1.6 neurons/mm2 of gray matter, respectively). Fibrosis thickness at the myelomeningocele scar level was reduced in UC-MSCs group (1269 µm vs 2624 µm). No tumors were observed. CONCLUSION: Fetal repair of myelomeningocele using allogenic UC-MSCs patch provides a moderate improvement in neurological functions, gray matter and neuronal preservation and prevented from fibrosis development at the myelomeningocele scar level.
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Meningomielocele , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Animales , Feto , Meningomielocele/terapia , Ovinos , Cordón UmbilicalRESUMEN
Sorafenib, an orally active multi-kinase inhibitor approved for the treatment of hepatocellular carcinoma (HCC), is primarily metabolized both via cytochrome P450 3A4 isoform (CYP3A4) and UGT1A9. Due to the contribution of these two biotransformation pathways, sorafenib is considered to be less susceptible than other agents to CYP3A4 drug-drug interactions. This report discusses a clinically relevant pharmacokinetic CYP3A4 drug-drug interaction between sorafenib and felodipine in an 80-year-old Caucasian patient with HCC. On day 15, after the introduction of sorafenib (400 mg bid), sorafenib plasma concentration was at 3.6 mg/L. Felodipine (5 mg bid), an anti-hypertensive agent that is exclusively CYP3A4 substrate, was then introduced due to grade 2 sorafenib-related hypertension. On day 30, hypertension was well controlled. However, sorafenib plasma concentration was 3-fold greater (11.4 mg/L) and the patient experienced grade-3 anorexia. Since neither diarrhea nor cutaneous side effects were noticed at this time, sorafenib treatment was continued at the same daily dosage. On day 45, sorafenib plasma concentration was stable (10.8 mg/L) before declining on days 60 and 75 (7.0 mg/L and 7.4 mg/L, respectively), which was probably related to an occurrence of grade-2 diarrhea. This observation suggests a pharmacokinetic interaction involving CYP3A4 inhibition by felodipine. According to the Drug Interaction Probability Scale, this interaction was possible. Since hypertension is a common toxicity of sorafenib, clinicians should be aware of this possible interaction. The clinical relevance of pharmacokinetic interactions involving CYP3A4 inhibition in HCC patients receiving sorafenib is analyzed in this case report.
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Antineoplásicos/farmacocinética , Bencenosulfonatos/farmacocinética , Inhibidores del Citocromo P-450 CYP3A , Felodipino/farmacología , Piridinas/farmacocinética , Anciano de 80 o más Años , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Bencenosulfonatos/efectos adversos , Bencenosulfonatos/uso terapéutico , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Citocromo P-450 CYP3A/metabolismo , Interacciones Farmacológicas , Felodipino/uso terapéutico , Humanos , Hipertensión/inducido químicamente , Hipertensión/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Masculino , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Piridinas/efectos adversos , Piridinas/uso terapéutico , SorafenibRESUMEN
In esophageal pathologies, such as esophageal atresia, cancers, caustic burns, or post-operative stenosis, esophageal replacement is performed by using parts of the gastrointestinal tract to restore nutritional autonomy. However, this surgical procedure most often does not lead to complete functional recovery and is instead associated with many complications resulting in a decrease in the quality of life and survival rate. Esophageal tissue engineering (ETE) aims at repairing the defective esophagus and is considered as a promising therapeutic alternative. Noteworthy progress has recently been made in the ETE research area but strong challenges remain to replicate the structural and functional integrity of the esophagus with the approaches currently being developed. Within this context, 3D bioprinting is emerging as a new technology to facilitate the patterning of both cellular and acellular bioinks into well-organized 3D functional structures. Here, we present a comprehensive overview of the recent advances in tissue engineering for esophageal reconstruction with a specific focus on 3D bioprinting approaches in ETE. Current biofabrication techniques and bioink features are highlighted, and these are discussed in view of the complexity of the native esophagus that the designed substitute needs to replace. Finally, perspectives on recent strategies for fabricating other tubular organ substitutes via 3D bioprinting are discussed briefly for their potential in ETE applications.
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Bioimpresión , Esófago/cirugía , Impresión Tridimensional , Calidad de Vida , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
BACKGROUND: Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) revealed their key role in immune regulation, offering promising therapeutic perspectives for immune and inflammatory diseases. We aimed to develop a production process of an UC-MSC-based product and then to characterize UC-MSC properties and immunomodulatory activities in vitro, related to their clinical use and finally, to transfer this technology to a good manufacturing practice (GMP) compliant facility, to manufacture an advanced therapy medicinal product (ATMP). METHODS: Fifteen human umbilical cords (UCs) were collected to develop the production process. Three batches of UC-MSCs from a single donor were characterized at basal state and after in vitro pro-inflammatory stimulation by interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα). Proliferation, immunophenotype, activation markers' expression and the inhibition of T cell proliferation were assessed. Finally, this technology was transferred to a GMP-compliant facility to manufacture an UC-MSC-based ATMP, from a single donor, using the explant method followed by the establishment of master and work cell stocks. RESULTS: Twelve UCs were processed successfully allowing to isolate UC-MSCs with doubling time and population doubling remaining stable until passage 4. CD90, CD105, CD73, CD44, CD29, CD166 expression was positive; CD14, CD45, CD31, HLA-DR, CD40, CD80 and CD86 expression was negative, while CD146 and HLA-ABC expression was heterogeneous. Cell morphology, proliferation and immunophenotype were not modified by inflammatory treatment. Indoleamine 2,3-dioxygenase (IDO) expression was significantly induced by IFNγ and IFNγ + TNFα versus non-treated cells. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression was induced significantly after priming. T cell proliferation was significantly decreased in the presence of UC-MSCs in a dose-dependent manner. This inhibitory effect was improved by IFNγ or IFNγ + TNFα, at UC-MSCs:PBMC ratio 1:10 and 1:30, whereas only IFNγ allowed to decrease significantly T cell proliferation at ratio 1:100. The manufacturing process of the UC-MSC-based ATMP was qualified and authorized by the French regulatory agency for clinical use (NCT04333368). CONCLUSION: This work allowed to develop an investigational UC-MSC-based ATMP authorized for clinical use. Our results showed that an inflammatory environment preserves the biological properties of UC-MSCs with an improvement of their immunomodulatory functions.
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Leucocitos Mononucleares , Células Madre Mesenquimatosas , Proliferación Celular , Células Cultivadas , Humanos , Inmunomodulación , Cordón UmbilicalRESUMEN
The fate of hematopoietic stem and progenitor cells (HSPCs) is regulated by their interaction with stromal cells in the bone marrow. However, the cellular mechanisms regulating HSPC interaction with these cells and their potential impact on HSPC polarity are still poorly understood. Here we evaluated the impact of cell-cell contacts with osteoblasts or endothelial cells on the polarity of HSPC. We found that an HSPC can form a discrete contact site that leads to the extensive polarization of its cytoskeleton architecture. Notably, the centrosome was located in proximity to the contact site. The capacity of HSPCs to polarize in contact with stromal cells of the bone marrow appeared to be specific, as it was not observed in primary lymphoid or myeloid cells or in HSPCs in contact with skin fibroblasts. The receptors ICAM, VCAM, and SDF1 were identified in the polarizing contact. Only SDF1 was independently capable of inducing the polarization of the centrosome-microtubule network.
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Médula Ósea/metabolismo , Médula Ósea/fisiología , Quimiocina CXCL12/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , HumanosRESUMEN
BACKGROUND: Mesenchymal stem/stromal cells (MSC) have immunomodulatory properties, studied in a wide range of diseases. Validated quality controls must confirm this activity in the context of clinical trials. This study presents a method's validation, assessing MSC's ability to inhibit lymphocyte proliferation, according to the ICH Q2 standard. METHODS: MSC were co-cultured with CellTrace™ Violet-labeled Peripheral blood mononuclear cells (PBMC) coming from a bank of ten donors, at seven different ratios for 7 days. Cell trace violet PBMC bank was validated in parallel. Flow cytometry analysis was used to obtain the division percentage of T cells. The percentage of inhibition of lymphocyte proliferation by MSC, for each ratio X, was calculated using the formula: Ratio × percentage of inhibition = (control percentage of division-ratio × percentage of division)/control percentage of division. The inhibition percentage of lymphocyte proliferation function of co-culture ratios was represented in a line graph. The corresponding area under the curve was calculated, representing MSC's ability to inhibit lymphocyte proliferation. RESULTS: Two cell trace violet PBMC banks were compared for bank validation. When compared using four different MSC samples coming each from a different donor, their area under the curve did not show any statistical differences and were correlated. Moreover, the stability of one cell trace violet PBMC bank was confirmed up to 509 days of storage. Analytical parameters were investigated for method validation. Analysis of repeatability and reproducibility respectively showed a standard deviation of 6.1% and 4.6%. The assay was robust regarding PBMC, as no statistical differences were found between inhibitory activities when testing three adjacent concentrations of PBMC. Still, attention is needed on MSC quantity as it can influence results. Linearity was evaluated: the percentage of inhibition of lymphocyte proliferation function of co-culture ratios was linear on the exploited range. Finally, the assay measurement range allowed to differentiate MSC presenting different inhibition activities. CONCLUSION: This quantification method displayed low analytical variability and no inter-bank variability of PBMC. However, MSC quantification should be checked before co-culture to reduce variability. Therefore, it could be used for the qualification of MSC batches' immunomodulatory activity.
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Leucocitos Mononucleares , Células Madre Mesenquimatosas , Proliferación Celular , Células Cultivadas , Citometría de Flujo , Prueba de Cultivo Mixto de Linfocitos , Reproducibilidad de los ResultadosAsunto(s)
Quirófanos , Humanos , Médula Ósea , Masculino , Femenino , Células de la Médula Ósea , Trasplante de Médula Ósea/métodosRESUMEN
In pathologies of the esophagus such as esophageal atresia, cancers, and caustic injuries, methods for full thickness esophageal replacement require the sacrifice of healthy intra-abdominal organs such as the stomach and the colon and are associated with high morbidity, mortality, and poor functional results. To overcome these problems, tissue engineering methods are developed to create a substitute with scaffolds and cells. The aim of this study was to develop a simple and safe decellularization process in order to obtain a clinical grade esophageal extracellular matrix. Following the decontamination step, porcine esophagi were decellularized in a bioreactor with sodium dodecyl sulfate and ethylenediaminetetraacetic acid for 3 days and were rinsed with deionized water. DNA was eliminated by a 3-hr DNase treatment. To remove any residual detergent, the matrix was then incubated with an absorbing resin. The resulting porcine esophageal matrix was characterized by the assessment of the efficiency of the decellularization process (DNA quantification), evaluation of sterility and absence of cytotoxicity, and its composition and biomechanical properties, as well as the possibility to be reseeded with mesenchymal stem cells. Complete decellularization with the preservation of the general structure, composition, and biomechanical properties of the native esophageal matrix was obtained. Sterility was maintained throughout the process, and the matrix showed no cytotoxicity. The resulting matrix met clinical grade criteria and was successfully reseeded with mesenchymal stem cells..
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Esófago/química , Matriz Extracelular/química , Ensayo de Materiales , Células Madre Mesenquimatosas/metabolismo , Ingeniería de Tejidos , Andamios del Tejido/química , Animales , Células Madre Mesenquimatosas/citología , PorcinosRESUMEN
BACKGROUND: Expansion protocols aim at both increasing the number of umbilical cord blood (UCB) hematopoietic stem cells and progenitor cells (HSPCs) and reducing the period of neutropenia in UCB HSPC graft. Because glycosaminoglycans (GAGs) are known to be important components of the hematopoietic niche and to modulate growth factor effects, we explored the use of GAG mimetic OTR4131 to potentiate HSPC's in vitro expansion and in vivo engraftment. METHODS: UCB CD34+ cells were expanded with serum-free medium, SCF, TPO, FLT3-lig and G-CSF during 12 days in the absence or the presence of increasing OTR4131 concentrations (0-100 µg/mL). Proliferation ratio, cell viability and phenotype, functional assays, migration capacity and NOD-scid/γc(-/-) mice engraftment were assessed after expansion. RESULTS: At Day 12, ratios of cell expansion were not significantly increased by OTR4131 treatment. Better total nucleated cell viability was observed with the use of 1 µg/mL GAG mimetic compared to control (89.6 % ± 3.7 % and 79.9 % ± 3.3 %, respectively). Phenotype analysis showed a decrease of monocyte lineage in the presence of OTR4131 and HSPC migration capacity was diminished when GAG mimetic was used at 10 µg/mL (10.9 % ± 4.1 % vs. 52.9 % ± 17.9 % for control). HSPC clonogenic capacities were similar whatever the culture conditions. Finally, in vivo experiments revealed that mice successfully engrafted in all conditions, even if some differences were observed during the first month. Three months after graft, bone marrow chimerism and blood subpopulations were similar in both groups. CONCLUSIONS: UCB HSPCs ex-vivo expansion in the presence of OTR4131 is a safe approach that did not modify cell function and engraftment capacities. In our experimental conditions, the use of a GAG mimetic did not, however, allow increasing cell expansion or optimizing their in vivo engraftment.