Number Of Transplanted Cells Research Articles

Deep learning-enabled quantification of simultaneous PET/MRI for cell transplantation monitoring

Current methods of invivo imaging islet cell transplants for diabetes using magnetic resonance imaging (MRI) are limited by their low sensitivity. Simultaneous positron emission tomography (PET)/MRI has greater sensitivity and ability to visualize cell metabolism. However, this dual-modality tool currently faces two major challenges for monitoring cells. Primarily, the dynamic conditions of PET such as signal decay and spatiotemporal change in radioactivity prevent accurate quantification of the transplanted cell number. In addition, selection bias from different radiologists renders human error in segmentation. This calls for the development of artificial intelligence algorithms for the automated analysis of PET/MRI of cell transplantations. Here, we combined K-means++ for segmentation with a convolutional neural network to predict radioactivity in cell-transplanted mouse models. This study provides a tool combining machine learning with a deep learning algorithm for monitoring islet cell transplantation through PET/MRI. It also unlocks a dynamic approach to automated segmentation and quantification of radioactivity in PET/MRI.

Expression analysis of genes involved in the expansion of hematopoietic stem cells (SCF, Flt3-L, TPO, IL-3, and IL-6) in unrestricted somatic stem cells cultured on fibrin

Umbilical cord blood (UCB) transplantation is a promising therapeutic approach for patients lacking HLA-matched donors. A main limitation to the use of UCB-derived HSCs (UCB–HSCs) is the low number of transplantable cells. Novel culture strategies are being developed to increase the number of HSCs. Unrestricted somatic stem cells (USSCs) have been identified as promising stromal cells for supporting HSC expansion. The current study aimed to explore the effect of fibrin on the expression of hematopoiesis-related genes (SCF, Flt3-L, TPO, IL-3, and IL-6) in USSCs. USSCs were isolated from UCB and characterized by flow cytometry and in vitro multilineage differentiation ability. DAPI staining and the MTT assay were used to assess the effect of fibrin on USSC viability. The cell attachment was evaluated using SEM. qRT-PCR was performed to evaluate the expression of SCF, Flt3-L, TPO, IL-3, and IL-6 in USSCs cultured on 3D fibrin scaffolds. USSCs were positive for CD73, CD105, and CD166 and negative for CD45. Alizarin red and Oil red O stains confirmed calcium deposition and lipid vacuoles in USSCs. Results obtained from DAPI and MTT assays revealed a positive effect of fibrin on USSC viability. Cells cultured on fibrin express significantly higher levels of SCF and TPO compared to those grown in a 2D environment. The positive effect of fibrin on IL-6 levels was reversed. Fibrin did not affect Flt3-L expression and IL-3 mRNA expression was not detected in either group. The results of this study provide the basis for developing further research on the ex vivo expansion of HSCs with USSCs.

Efficiency assessment of irrigation as an alternative method for improving the regenerative potential of non-healing wounds.

The application of mesenchymal stem/stromal cells (MSC) in regenerative medicine offers hope for the effective treatment of incurable or difficult‐to‐heal diseases. However, it requires the development of unified protocols for both safe and efficient cell acquisition and clinical usage. The therapeutic effect of fat grafts (containing stem cells) in non‐healing wounds has been discussed in previous studies, although the application requires local or general anaesthesia. The treatment of MSC derived from adipose tissue (ASC) could be a less invasive method, and efficient delivery could lead to more favourable outcomes, which should encourage clinicians to use such therapeutic approaches more frequently. Therefore, the aim of this study was to optimise the methods of ASC isolation, culture and administration while maintaining their high survival, proliferation and colonisation potential. The ASC were isolated by an enzymatic method and were characterised according to International Society for Cellular Therapy and International Federation for Adipose Therapeutics and Science guidelines. To assess the opportunity to obtain a sufficient number of cells for transplantation, long‐term cell cultures in two oxygen concentrations (5% vs. 21%) were conducted. For these cultures, the population doubling time, the cumulative time for cell population doublings and the rate of cell senescence were estimated. In a developed and pre‐defined protocol, ASC can be efficiently cultured at physiological oxygen concentrations (5%), which leads to faster proliferation and slower cell senescence. Subsequently, to select the optimal and minimally invasive methods of ASC transplantation, direct cell application with an irrigator or with skin dressings was analysed. Our results confirmed that both the presented methods of cell application allow for the safe delivery of isolated ASC into wounds without losing their vitality. Cells propagated in the described conditions and applied in non‐invasive cell application (with an irrigation system and dressings) to treat chronic wounds can be a potential alternative or supplement to more invasive clinical approaches.

Towards the standardization of methods of tissue processing for the isolation of mesenchymal stromal cells for clinical use

Multipotent mesenchymal stromal cells (MSCs) are currently the most extensively studied type of adult stem cells in advanced stages of development in the field of regenerative medicine. The biological properties of MSCs have generated great hope for their therapeutic use in degenerative and autoimmune conditions that, at present, lack effective treatment options. Over the last decades, MSCs have been typically obtained from adult bone marrow, but the extraction process is highly invasive and the quality and numbers of isolated cells is drastically influenced by patient age, medication and associated comorbidities. Therefore, there is currently an open discussion on the convenience of allogeneic over autologous treatments, despite potential disadvantages such as rejection by the host. This shift to the allogeneic setting entails the need for high production of MSCs to ensure availability of sufficient cell numbers for transplantation, and therefore making the search for alternative tissue sources of highly proliferative MSC cultures with low levels of senescence occurrence, which is one of the greatest current challenges in the scale up of therapeutic cell bioprocessing. Herein we (i) present the main isolation protocols of MSCs from bone marrow, adipose tissue and Wharton’s jelly of the umbilical cord; and (ii) compare their qualities from a bioprocess standpoint, addressing both quality and regulatory aspects, in view of their anticipated clinical use.

Stem cell therapy for osteonecrosis of femoral head: Opportunities and challenges.

Osteonecrosis of the femoral head (ONFH) is a progressive disease with a complex etiology and unclear pathogenesis, resulting in severe hip pain and dysfunction mainly observed in young patients. Although total hip arthroplasty (THA) is the most effective treatment for patients with ONFH in the terminal stage, the results of THA in young patients or active populations are often not favorable, with some complications related to the prosthesis. With the development of biotechnology, an increasing number of studies pay attention to use of stem cells for the treatment of ONFH. Stem cells are characterized by the ability to self-renew and differentiate into multiple cell types, including differentiation into osteoblasts and endothelial cells to mediate bone repair and angiogenesis. Furthermore, stem cells can offer growth factors to promote blood supply in the necrotic regions by paracrine effects. Therefore, stem cell therapy has become one of the hip-preserving alternatives for ONFH. This review summarized the current trends in stem cell therapy for ONFH, from clinical applications to related basic research, and showed that an increasing number of studies have confirmed the effectiveness of stem cell therapy in ONFH. However, many unsolved problems and challenges in practical applications of stem cell therapy still exist, such as patient selection, standardized procedures, safety assessment, and the fate of transplanted cells in the body. Additional studies are required to find ideal cell sources, appropriate transplantation methods, and the optimal number of cells for transplantation.

Generation of Insulin-Producing Cells from Canine Adipose Tissue-Derived Mesenchymal Stem Cells.

The potential of mesenchymal stem cells (MSCs) to differentiate into nonmesodermal cells such as pancreatic beta cells has been reported. New cell-based therapy using MSCs for diabetes mellitus is anticipated as an alternative treatment option to insulin injection or islet transplantation in both human and veterinary medicine. Several protocols were reported for differentiation of MSCs into insulin-producing cells (IPCs), but no studies have reported IPCs generated from canine MSCs. The purpose of this study was to generate IPCs from canine adipose tissue-derived MSCs (AT-MSCs) in vitro and to investigate the effects of IPC transplantation on diabetic mice in vivo. Culturing AT-MSCs with the differentiation protocol under a two-dimensional culture system did not produce IPCs. However, spheroid-like small clusters consisting of canine AT-MSCs and human recombinant peptide μ-pieces developed under a three-dimensional (3D) culture system were successfully differentiated into IPCs. The generated IPCs under 3D culture condition were stained with dithizone and anti-insulin antibody. Canine IPCs also showed gene expression typical for pancreatic beta cells and increased insulin secretion in response to glucose stimulation. The blood glucose levels in streptozotocin-induced diabetic mice were decreased after injection with the supernatant of canine IPCs, but the hyperglycemic states of diabetic mice were not improved after transplanting IPCs subcutaneously or intramesenterically. The histological examination showed that the transplanted small clusters of IPCs were successfully engrafted to the mice and included cells positive for insulin by immunofluorescence. Several factors, such as the transplanted cell number, the origin of AT-MSCs, and the differentiation protocol, were considered potential reasons for the inability to improve the hyperglycemic state after IPC transplantation. These findings suggest that canine AT-MSCs can be differentiated into IPCs under a 3D culture system and IPC transplantation may be a new treatment option for dogs with diabetes mellitus.

Long-Term Survival of Transplanted Autologous Canine Liver Organoids in a COMMD1-Deficient Dog Model of Metabolic Liver Disease.

The shortage of liver organ donors is increasing and the need for viable alternatives is urgent. Liver cell (hepatocyte) transplantation may be a less invasive treatment compared with liver transplantation. Unfortunately, hepatocytes cannot be expanded in vitro, and allogenic cell transplantation requires long-term immunosuppression. Organoid-derived adult liver stem cells can be cultured indefinitely to create sufficient cell numbers for transplantation, and they are amenable to gene correction. This study provides preclinical proof of concept of the potential of cell transplantation in a large animal model of inherited copper toxicosis, such as Wilson’s disease, a Mendelian disorder that causes toxic copper accumulation in the liver. Hepatic progenitors from five COMMD1-deficient dogs were isolated and cultured using the 3D organoid culture system. After genetic restoration of COMMD1 expression, the organoid-derived hepatocyte-like cells were safely delivered as repeated autologous transplantations via the portal vein. Although engraftment and repopulation percentages were low, the cells survived in the liver for up to two years post-transplantation. The low engraftment was in line with a lack of functional recovery regarding copper excretion. This preclinical study confirms the survival of genetically corrected autologous organoid-derived hepatocyte-like cells in vivo and warrants further optimization of organoid engraftment and functional recovery in a large animal model of human liver disease.

Engraftable Induced Pluripotent Stem Cell-Derived Neural Precursors for Brain Repair.

Stem cell transplantation has attracted great interest for treatment of neurodegenerative diseases to provide neuroprotection, repair the lesioned neuronal network and restore functionality. Parkinson's disease (PD), in particular, has been a preferred target because motor disability that constitutes a core pathology of the disease is associated with local loss of dopaminergic neurons in a specific brain area, the substantia nigra pars compacta. These cells project to the striatum where they deliver the neurotransmitter dopamine that is involved in control of many aspects of motor behavior. Therefore, cell transplantation approaches in PD aim to replenish dopamine deficiency in the striatum. A major challenge in developing cell therapy approaches is the ability to generate large numbers of transplantable cells in a reliable and reproducible manner. In recent years the technological breakthrough of induced pluripotent stem cells (iPSCs) has demonstrated that this is possible at a preclinical level, accelerating clinical translation. A second important issue is to efficiently differentiate iPSCs into dopaminergic neuronal progenitors with restricted proliferation potential in order to avoid cellular overgrowth in vivo and minimize the risk of tumorigenesis. Here we describe an effective protocol that includes human iPSC differentiation to the dopaminergic lineage and enrichment in neuronal precursor cells expressing the polysialylated form of the neural cell adhesion molecule PSA-NCAM, through magnetically activated cell sorting. The resulting cells are transplanted and shown to survive, differentiate, and integrate within a striatal lesion model generated by unilateral 6-hydroxydopamine administration in mice of the NOD/SCID strain that supports xenografts.

Salidroside enhances proliferation and maintains phenotype of articular chondrocytes for autologous chondrocyte implantation (ACI) via TGF-β/Smad3 Signal

Autologous chondrocyte implantation (ACI) is commonly used for the treatment of cartilage defects. Since the cell number for transplantation is limited, the expand culture of chondrocytes in vitro is needed. However, the phenotype of chondrocytes is easy to lose in monolayer cultured in vitro. Traditional growth factors such as transformation growth factor -β1 (TGF-β1) have been used for promoting the proliferation and maintained the phenotype of chondrocytes, but the high cost and functional heterogeneity limit their clinical application. It is of significant to develop substitutes that can accelerate proliferation and prevent dedifferentiation of chondrocytes for further study. In our present study, the effect of salidroside on proliferation and phenotype maintenance of chondrocytes and cartilage repair was investigated by performing the cell viability, morphology, glycosaminoglycan (GAG) synthesis, cartilage relative genes expression, macroscopic and histological analyzsis. The TGF-β/smad3 signal which may involve in the protective effect of salidroside on chondrocytes was also detected by ELISA and qRT-PCR assays. The results indicated that salidroside could promote chondrocytes proliferation and enhance synthesis of cartilage extracellular matrix (ECM). Expression of collagen type I was significantly down-regulated which suggesting that salidroside could prevent chondrocytes from dedifferentiation. The in vivo experiments for cartilage repair also indicated that in the treatment of salidroside, chondrocytes used for ACI significantly accelerated the hyaline cartilage repair. While in the absence of salidroside, the repaired cartilage is mainly the fibrous cartilage. Additional experiments demonstrated that salidroside promotes the proliferation and maintain the phenotype of chondrocytes by activate the TGF-β/smad3 signal. Salidroside may be a potential agent for ACI to promote the proliferation and maintain the phenotype of chondrocytes expansion in vitro.

Molecular Mechanisms Responsible for Therapeutic Potential of Mesenchymal Stem Cell-Derived Secretome.

Mesenchymal stem cell (MSC)-sourced secretome, defined as the set of MSC-derived bioactive factors (soluble proteins, nucleic acids, lipids and extracellular vesicles), showed therapeutic effects similar to those observed after transplantation of MSCs. MSC-derived secretome may bypass many side effects of MSC-based therapy, including unwanted differentiation of engrafted MSCs. In contrast to MSCs which had to be expanded in culture to reach optimal cell number for transplantation, MSC-sourced secretome is immediately available for treatment of acute conditions, including fulminant hepatitis, cerebral ischemia and myocardial infarction. Additionally, MSC-derived secretome could be massively produced from commercially available cell lines avoiding invasive cell collection procedure. In this review article we emphasized molecular and cellular mechanisms that were responsible for beneficial effects of MSC-derived secretomes in the treatment of degenerative and inflammatory diseases of hepatobiliary, respiratory, musculoskeletal, gastrointestinal, cardiovascular and nervous system. Results obtained in a large number of studies suggested that administration of MSC-derived secretomes represents a new, cell-free therapeutic approach for attenuation of inflammatory and degenerative diseases. Therapeutic effects of MSC-sourced secretomes relied on their capacity to deliver genetic material, growth and immunomodulatory factors to the target cells enabling activation of anti-apoptotic and pro-survival pathways that resulted in tissue repair and regeneration.

Phenotypic and Functional Equivalency of Digested Bone Marrow Mesenchymal Stem Cells to Aspirated Bone Marrow Mesenchymal Stem Cells

Adult mesenchymal stem cells (MSCs) are found throughout the body, including in the dental pulp, adipose tissue, and bone marrow (BM), among other tissues. MSCs are receiving increased attention in the medical field due to their therapeutic and regenerative effects. However, since MSCs comprise only 0.01–0.001% of BM cells, traditional methods of living donor aspiration yield a few hundred to a few thousand cells which must be extensively expanded to obtain a clinically significant number of transplantable cells. Results from late‐stage clinical trials suggest that MSC lose functional potency with expansion; thus, there is a need for large sources of primary MSCs for therapeutic use. BM from deceased donors are a potential source of large quantities of unpassaged MSCs. However, the functional equivalency of deceased donor‐ to living donor‐derived MSC has not been previously established. This study compared phenotypic and functional properties of enzymatically digested MSCs from deceased donor vertebral bodies (VBs) to MSCs obtained from aspirates of living BM.MSCs isolated from living and deceased donors were characterized according to the International Society for Cellular Therapy (ISCT)‐criteria. VBs obtained under IRB approval from five deceased organ donors were ground and filtered to extract BM. The remaining cortical and trabecular bone fragments were digested using a mix of collagenase and neutral protease (DE10, Vitacyte, Indianapolis, IN) to obtain MSCs. Digested and whole BM mononuclear cells were then plated on uncoated plastic and plastic‐adherent MSCs were expanded through four passages. Three different aspirated BM MSC samples from LaCell, L.L.C. were used as live donor controls and were expanded through three passages. MSCs were characterized at each passage for adipogenic, chondrogenic, and osteogenic differentiation potential, CFU‐F, population doubling time, and flow cytometry.Both living aspirate and deceased digest MSCs showed fibroblast‐like phenotypes at each passage during expansion. Both also differentiated into adipocytes, chondrocytes, and osteocytes. Flow cytometry revealed both living aspirate and deceased digest samples were negative for CD14, CD19, CD34, CD45, and HLA‐DR and were positive for CD73, CD90, and CD105. There was no significant difference in viability between LaCell's aspirated sample and MSCs from digested VBs. Both living and deceased donor derived MSCs were capable of forming colonies further demonstrating their equivalency.The results of these experiments show that deceased donor digested MSCs are comparable to living donor aspirated MSCs in their phenotype, tri‐lineage differentiation capacity, viability, and cell surface markers. Additionally, deceased donor digested MSCs are superior to living donor MSCs in their less intrusive recovery procedures and greater potential yield, given that there are no restrictions on the amount of BM harvested to reduce donor morbidity.Support or Funding InformationResearch was supported by:A grant by the National Institutes of Health (NIAID Grant No. 1 U01 AI138334‐01)Ossium Health, IncMarian College of Osteopathic MedicineThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High throughput screening reveals no significant changes in protein synthesis, processing, and degradation machinery during passaging of mesenchymal stem cells 1.

Increasing reports of successful and safe application of bone marrow derived mesenchymal stem cells (BM-MSCs) for cell therapy are pouring in from numerous studies. However poor survival of transplanted cells in the recipient has impaired the benefits of BM-MSCs based therapies. Therefore cell product preparation procedures pertaining to MSC therapy need to be optimized to improve the survival of transplanted cells. One of the important ex vivo procedures in the preparation of cells for therapy is passaging of BM-MSCs to ensure a suitable number of cells for transplantation, which may affect the turnover of proteins involved in regulation of cell survival and (or) death pathways. In the current study, we investigated the effect of an increase in passage number of BM-MSCs in cell culture on the intracellular protein turnover (protein synthesis, processing, and degradation machinery). We performed proteomic analysis of BM-MSCs at different passages. There was no significant difference observed in the ribosomal, protein processing, and proteasomal pathways related proteins in BM-MSCs with an increase in passage number from P3 to P7. Therefore, expansion of MSCs in the cell culture in clinically relevant passages (Passage 3-7) does not affect the quality of MSCs in terms of intracellular protein synthesis and turnover.

Isolation of Rat Olfactory Ensheathing Cells and Their Use in the Therapy of Posttraumatic Cysts of the Spinal Cord.

We evaluated the efficacy of rat olfactory ensheathing cells in the therapy of experimental cysts of the spinal cord. Improvement of the motor function of the hind limbs after transplantation of the olfactory ensheathing cells into the posttraumatic spinal cord cysts rats was found. We also determined the required number of cells for transplantation and demonstrated a neuroprotective effect of this dosage. For further clinical studies, autologous tissue-specific cell preparation of olfactory ensheathing cells has to be created. Cell therapy in combination surgical and pharmacological treatment will substantially improve the quality of life of patients with posttraumatic spinal cord cysts.

Stem cell-based therapies for neurological disorders

Cell-based therapies have been previously performed using fetal tissues for some central nervous system (CNS) disorders, such as Parkinson’s disease. However, it can be difficult to collect a large number of cells for transplantation. Recent studies revealed that some stem cells can act as potential sources of cell-based therapies for degenerative and damaged areas in the CNS. In addition, stem cells can be used as cellular delivery vehicles for brain tumor because of tumor-tropic migratory capacity. Embryonic stem (ES) cells, mesenchymal stem cells (MSCs), and induced pluripotent stem (iPS) cells are the most attractive stem cells. iPS cells can be efficiently differentiated to neural stem cells and have the possibilities to overcome the ethical issues associated with ES cells. Therefore, cell-based therapies using iPS cells can be developed specifically for neurological disorders. In this article, we review the characteristics of ES cells, MSCs, and iPS cells as cell sources for stem cell-based therapies, and then discuss preclinical data and ongoing clinical trials for the CNS disorders.

Materials-Directed Differentiation of Mesenchymal Stem Cells for Tissue Engineering and Regeneration.

Cell-based therapies are a promising alternative to grafts and organ transplantation for treating tissue loss or damage due to trauma, malfunction, or disease. Over the past two decades, mesenchymal stem cells (MSCs) have attracted much attention as a potential cell population for use in regenerative medicine. While the proliferative capacity and multilineage potential of MSCs provide an opportunity to generate clinically relevant numbers of transplantable cells, their use in tissue regenerative applications has met with relatively limited success to date apart from secreting paracrine-acting factors to modulate the defect microenvironment. Presently, there is significant effort to engineer the biophysical properties of biomaterials to direct MSC differentiation and further expand on the potential of MSCs in tissue engineering, regeneration, and repair. Biomaterials can dictate MSC differentiation by modulating features of the substrate including composition, mechanical properties, porosity, and topography. The purpose of this review is to highlight recent approaches for guiding MSC fate using biomaterials and provide a description of the underlying characteristics that promote differentiation toward a desired phenotype.

Human CD34+ Cells from Different Sources Disclose a Specific Stemness Signature

Over the past decades outcomes of clinical hematopoietic stem cell transplants have established a clear relationship between the sources of hematopoietic stem cells (HSCs) infused and their differential homing and engraftment properties. For a long time, bone marrow (BM) harvest has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution following myeloablative conditioning regimen. At present, mobilized peripheral blood (PB) is commonly used for hematopoietic cells transplantation in both adults and children, particularly in the autologous setting, and it has progressively replaced BM as the source of HSCs.HSCs are maintained in their niche by binding to cellular determinants through adhesion molecules and diverse strategies are currently used to promote their egress from BM to PB. Traditionally, the growth factor granulocyte-colony stimulating factor (G-CSF) represents the gold standard agent to mobilize HSPCs for transplantation. Nevertheless, other compounds have been recently tested.One of the most successful mobilizing agents is Plerixafor (AMD3100, Mozobil™), a bicyclam molecule that selectively and reversibly antagonizes the binding of stromal cell derived factor-1 (SDF-1), located on the surface of BM stromal cells and osteoclasts, to chemokine CXC-receptor-4 (CXCR4), located on the surface of HSPCs, with the subsequent mobilization in the blood. The use of this drug is currently approved by FDA and EMA in combination with G-CSF, in patients affected by lymphoma or multiple myeloma whose cells mobilize poorly with G-CSF alone. Clinical trials demonstrated that Plerixafor alone safely and rapidly mobilizes HSCs also in healthy donors, beta-thalassemia patients and pediatric patients affected by malignancies. Previous characterization studies on non-human primates and human samples of Plerixafor mobilized cells in comparison to cells mobilized by G-CSF alone or in combination with Plerixafor showed a different expression profile, cell composition and engrafting potential in a xenotransplant model. From these studies remains unsolved whether Plerixafor, G-CSF, or their combination mobilizes different primitive HSC populations, defined both by multimarker immunophenotype and in vivo functional analysis.In the present study we investigated by controlled comparative analysis the functional and molecular hallmarks of human HSCs collected from BM, G-CSF and/or Plerixafor mobilized peripheral blood. We show that Plerixafor alone mobilizes preferentially long-term hematopoietic stem cells (LT-HSCs), defined as CD34+CD38/lowCD90+CD45RA-CD49f+ cells and primitive populations of HSCs. These cells possess higher ability to home to hematopoietic niches and engraft in NOD/SCID/IL2rγnull (NSG) mice, resulting in enriched scid-repopulating cell frequency, in comparison to other sources. The higher content of CXCR4+ and CD49f+ cells correlates with this feature. Furthermore, global gene expression profiling highlights the superior in vivo reconstitution activity of Plerixafor mobilized cells. The "stemness" signature of cells dislodged from their niche by the drug is attenuated by the combined use with G-CSF, which emphasizes the gene expression profile induced by G-CSF treatment.These data indicate that a qualitative advantage accounts for the superior performance of Plerixafor mobilized cells. These findings provide the rationale for using a suboptimal dose of more primitive HSCs when target cell number for transplantation is limited, or when G-CSF mobilization is too risky like in sickle cell anemia patients.Moreover, CD34+ cells mobilized by Plerixafor alone or with the combination of G-CSF are efficiently transduced by a lentiviral vector encoding for human ß-globin gene (GLOBE LV) and are able to engraft and differentiate in vivo, supporting their use for gene therapy applications. DisclosuresCiceri:MolMed SpA: Consultancy.

MEM α Promotes Cell Proliferation and Expression of Bone Marrow Derived Equine Mesenchymal Stem Cell Gene Markers but Depresses Differentiation Gene Markers

Equine bone marrow–derived mesenchymal stem cells (BM-eMSCs) have been used in veterinary clinics and research worldwide. For clinical use, in vitro propagation of BM-eMSCs is required (at least 2 weeks) to yield sufficient cell number for transplantation. Many culture media have been used to propagate human and animal MSCs but there are very few comparative studies of equine mesenchymal stem cells (eMSCs). The best culture media must promote cell proliferation and maintain eMSC properties. The objective of this study was to compare five basic commercial culture media by examining the proliferation rate and a representative MSC gene expression profile. Five culture media Dulbecco's modified eagle medium (DMEM)-LG, DMEM-HG, minimum essential medium alpha (MEM α), RPMI-1640, and DMEM/F12 were compared. Cultured BM-eMSCs were collected at day 7 and day 14 to measure cell number and gene expression. Relative gene expression was performed using real-time RT-PCR. Our results showed that MEM α was significantly superior to other media (P < .05) in 14-day culture, enhancing the expression of eMSC genes (ITGB1, CD44 and POU5F1) but depressing differentiation genes (DCN, PPARG and ADIPOQ) in addition to promoting rapid cell proliferation when compared to the other media. We concluded that MEM α was the best media to propagate BM-eMSCs up to 14 days as it supported cell proliferation while maintaining eMSC gene expression. From this, we propose that MEM α may be the most suitable medium for short-term culture of BM-eMSCs for cell transplantation studies.

Transient Tcf3 Gene Repression by TALE-Transcription Factor Targeting.

Transplantation of hematopoietic stem and progenitor cells (HSCs) i.e., self-renewing cells that retain multipotentiality, is now a widely performed therapy for many hematopoietic diseases. However, these cells are present in low number and are subject to replicative senescence after extraction; thus, the acquisition of sufficient numbers of cells for transplantation requires donors able to provide repetitive blood samples and/or methods of expanding cell numbers without disturbing cell multipotentiality. Previous studies have shown that HSCs maintain their multipotentiality and self-renewal activity if TCF3 transcription function is blocked under B cell differentiating conditions. Taking advantage of this finding to devise a new approach to HSC expansion in vitro, we constructed an episomal expression vector that specifically targets and transiently represses the TCF3 gene. This consisted of a vector encoding a transcription activator-like effector (TALE) fused to a Krüppel-associated box (KRAB) repressor. We showed that this TALE-KRAB vector repressed expression of an exogenous reporter gene in HEK293 and COS-7 cell lines and, more importantly, efficiently repressed endogenous TCF3 in a human B lymphoma cell line. These findings suggest that this vector can be used to maintain multipotentiality in HSC being subjected to a long-term expansion regimen prior to transplantation.

Optimizing autologous cell grafts to improve stem cell gene therapy.

Over the past decade, stem cell gene therapy has achieved unprecedented curative outcomes for several genetic disorders. Despite the unequivocal success, clinical gene therapy still faces challenges. Genetically engineered hematopoietic stem cells are particularly vulnerable to attenuation of their repopulating capacity once exposed to culture conditions, ultimately leading to low engraftment levels posttransplant. This becomes of particular importance when transduction rates are low or/and competitive transplant conditions are generated by reduced-intensity conditioning in the absence of a selective advantage of the transduced over the unmodified cells. These limitations could partially be overcome by introducing megadoses of genetically modified CD34(+) cells into conditioned patients or by transplanting hematopoietic stem cells hematopoietic stem cells with high engrafting and repopulating potential. On the basis of the lessons gained from cord blood transplantation, we summarize the most promising approaches to date of increasing either the numbers of hematopoietic stem cells for transplantation or/and their engraftability, as a platform toward the optimization of engineered stem cell grafts.

Mesenchymal Stem Cells Derived from Peripheral Blood Retain Their Pluripotency, but Undergo Senescence During Long-Term Culture.

Peripheral blood-derived mesenchymal stem cells (PB-MSCs) show promise as a source of cells for autologous transplantation because they can be harvested through minimally invasive procedures. To ensure adequate numbers of cells for transplantation and tissue regeneration, PB-MSCs must first be cultured and expanded in vitro, but whether long-term passage modifies their properties has been poorly understood. In this study we triggered production of PB-MSCs in rabbits using granulocyte colony-stimulating factor (G-CSF) and AMD3100, and then isolated and expanded the cells in culture until they reached a state of senescence, usually after about 20 passages. Cultures of low-, middle-, and high-passage numbers were compared in terms of morphology, proliferative capacity, phenotype, differentiation potential, apoptosis, metabolic indicators, and senescence. As passage number increased, MSCs retained their elongated spindle shape, but became larger and flatter, slowed in growth gradually, and increased proportion of cells showed G1 arrest. The proportions of apoptotic cells, production of reactive oxygen species (ROS), and ADP/ATP ratio increased with passage number. Expression of senescence-associated β-galactosidase increased, while telomerase activity decreased. On the other hand, cultures did not show significant changes in phenotype or lose their ability to differentiate into three lineages as passage number increased. These results suggest that PB-MSCs maintain their stem cell properties during prolonged culturing, but they undergo senescence that may be due to apoptosis and production of ROS. These findings may help to standardize in vitro production of PB-MSCs for tissue engineering.