High Proliferative Capacity Research Articles

CD4+CD28null T cells are expanded in moderately active systemic lupus erythematosus and secrete pro-inflammatory interferon gamma, depending on the Disease Activity Index.

Pathogenic CD4+CD28null cells are characterized by inflammatory cytokine synthesis and tropism to the inflamed tissues. Recent studies showed the involvement of CD28null T cells in a severe clinical outcome of lupus. However, their role in moderately active disease is still unresolved. We examined the levels of circulating CD4+CD28null cells and CD8+CD28null suppressor T cells. We also compared the CD4+CD28null and CD4+CD28+ T-cell functional properties, including the expression of interferon gamma (IFN-γ) and Ki67 among systemic lupus erythematosus (SLE) patients (n = 20) and healthy controls (n = 20). All the patients were under immunosuppressive treatment and exhibited moderate SLE activity (median SLE Disease Activity Index (SLEDAI) = 6). In patients, we found elevated CD4+CD28null and unchanged levels of suppressor CD8+CD28null T cells. There was no difference between patients and controls in IFN-γ and Ki67-expressing CD4+, CD4+CD28+, and CD4+CD28null T cells, except for higher IFN-γ levels in CD4+CD28+ T cells in SLE. In each studied group, we observed a higher preponderance of IFN-γ- and Ki67-expressing cells among CD4+CD28null T cells and lower levels of IFN-γ in CD4+CD28null T cells compared to the CD28+ subset. Similarly, Ki67 intensity was decreased in healthy CD4+CD28null cells, whereas in patients, comparably high expression was observed in both subsets. IFN-γ intensity in CD4+CD28null T cells correlated with SLEDAI. SLE with a moderately active clinical course is characterized by peripheral blood expansion of CD4+CD28null T cells and a normal abundance of suppressor CD8+CD28null T cells. The demonstration that these pathogenic CD4+ T cells, despite the lack of CD28, maintain the ability to produce pro-inflammatory IFN-γ positively correlated with disease activity as well as relatively high proliferative capacity may suggest their potentially predictive role in SLE flares.

A subpopulation of human Adark spermatogonia behaves as the reserve stem cell.

Human spermatogonial stem cells (SSCs) are an essential source to maintain spermatogenesis as an efficient process for daily sperm production with high self-renewal capacity along adulthood. However, the phenotype and the subpopulation that represent the real reserve SSC for the human testis remain unknown. Moreover, although SSC markers have been described for undifferentiated spermatogonia (Adark and Apale), the existence of a specific subtype that could be identified as the actual/true SSC has not yet been fully determined. Herein we evaluated spermatogonial morphology, kinetics, positioning regarding blood vasculature in relation to protein expression (UTF1, GFRA1, and KIT) as well as proliferative activity (MCM7) and identified a small subpopulation of Adark with nuclear rarefaction zone (AdVac) that behaves as the human reserve SSC. We show that AdVac is the smallest human spermatogonial population (10%), staying quiescent (89%) and positioned close to blood vessels throughout most of the stages of the seminiferous epithelium cycle (SEC) and divides only at stages I and II. Within this AdVac population, we found a smaller pool (2% of A undifferentiated spermatogonia) of entirely quiescent cells exhibiting a high expression of UTF1 and lacking GFRA1. This finding suggests them as the real human reserve SSC (AdVac UTF1+/GFRA1-/MCM7-). Additionally, Adark without nuclear vacuole (AdNoVac) and Apale have similar kinetic and high proliferative capacity throughout the SEC (47%), indicating that they are actively dividing undifferentiated spermatogonia. Identification of human stem cells with evident reserve SSC functionality may help further studies intending to sort SSCs to treat male diseases and infertility.

Anticancer mechanisms determination of L‐aminoacids compounds serie on MDA‐MB‐231 cell line TNBC

Breast cancer represents the second most frequently diagnosed neoplasm worldwide of which approximately 15% is subclassified as triple negative breast cancer (TNBC) [1], characterized by the absence of estrogen receptors, progesterone and growth factor Her2/neu. TNBC is considered as one of the most aggressive cancers, due to its null response to current endocrine therapies and trastuzumab [2]. Breast cancer is a pathology associated with aging and unhealthy lifestyles, it has been proven through in vitro studies that MDA‐MB‐231 TNBC cells overexpress HDAC8 as a consequence of epigenetic deregulation mechanisms, linked to the high proliferative capacity and therefore, a high risk of recurrence and low survival [3]. In this context, our work group have evaluated a serie of compounds on the MDA‐MB‐231 cell line, these compounds have been designed, synthesized and evaluated as iHDAC8 by Docking. Therefore, this work presents the cytotoxicity and evaluation of the antitumor effect on the MDA‐MB‐231 cell line of three compounds derived from L‐amino acids, BisLys, GluEt and I1, which contain the isoindolinyl group and I1 which has a carboxybenzoyl group. To evaluate citotoxicity of BisLys, GlutEt and I1 compounds, MDA‐MB‐231 cells were seeded and incubated at 37°C in a 5% CO2 with DMEM F12 medium supplemented with FBS. Once the cells reached 95% confluence they were treated for 24 hours adding 8 different concentrations of each of the compounds; DMEM F12 medium was used as a negative control and SAHA as a positive control. The proliferation and viability of TNBC cells was quantified by the colorimetric for 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Type of cell death whether it was necrosis or apoptosis was determined using Annexine V and propidium iodide by flow cytometry. Finally, the blocked cell cycle progression by those compounds was determined using Cayman 10009349 Kit by flow cytometry. The BisLys, GluEt and I1 compounds showed IC50 values of 0.83, 0.66 and I1 3.42 mM, respectively, and inhibited cell proliferation and viability in a dose‐dependent manner on the MDA‐MB‐231 cell line of TNBC. The GluEt compound induced death mainly by necrosis and the BisLys and I1 compounds by apoptosis. The BisLys, GluEt and I1 compounds induced cell cycle arrest in a dose‐dependent manner at the expense of the subG0/G1 phase. The compounds that induced the greatest apoptosis in MDA‐MB‐231 cells were BisLys and I1 which makes them leading compounds in suppressing the growth of MDA‐MB‐231 TNBC cells.

Interaction Between Near-Infrared Radiation and Temozolomide in a Glioblastoma Multiform Cell Line: A Treatment Strategy?

Photodynamic therapy (PDT) is a potential therapeutic modality against cancer, resulting from the interaction of a photosensitizer (PS) and radiation that generates damage to tumor cells. The use of near-infrared radiation (IR-A) is relevant because presents recognized biological effects, such as antioxidant, neuroprotective and antitumor effects. Glioblastoma is the most aggressive central nervous system (CNS) neoplasm with high proliferation and tissue invasion capacity and is resistant to radio and chemotherapy. Here, we evaluated in vitro the possible interaction of temozolomide (TMZ) with IR-A in a glioblastoma cell line (C6) and in a human keratinocyte cell line (HaCat) how non-tumor cell model, in an attempt to search for a new treatment strategy. The effects of TMZ, IR-A and the interaction between TMZ and IR-A was evaluated by viability exclusion with trypan blue. To perform the interaction experiments, we have chosen 10μM TMZ and 4.5J/cm2 of IR-A. From this, we evaluated cytotoxicity, cell proliferation, intracellular reactive oxygen species levels (ROS), as well as the process of cell migration and the P-gp and MRP-1 activity. Cell death mainly due to apoptosis, followed by necrosis, decreased cell proliferation, increased ROS levels, decreased cell migration and decreased P-gp and MRP1 activity were observed only when there was interaction between TMZ and IR-A in the C6 cell line. The interaction between TMZ and IR-A was not able to affect cell proliferation in the HaCat non-tumor cell line. Our results suggest that this interaction could be a promising approach and that in the future may serve as an antitumor strategy for PDT application.

Biological responses of dental pulp cells to surfaces modified by collagen 1 and fibronectin.

Collagen 1 (COL1) and fibronectin (FN) are extracellular matrix proteins that contribute in cell activity and involve in regulating dental pulp cells (DPCs). The purpose of this study was to investigate the effect of COL1 and FN on the behavior of DPCs. Here, DPCs were grown under three different conditions: COL1 coating, FN coating, and control group without coating. The proliferation and differentiation of DPCs were investigated. DPCs in osteogenic media were able to differentiate into osteoblastic phenotype. The morphological analysis revealed no obvious difference on the shape of cells. Cells had spread well on both coated and noncoated culture plates with slightly more spreading in the coated plates after 24 hr. The MTT analysis did not demonstrate a significant difference at 1 and 3 hr among the groups, but interestingly, the analysis disclosed more cells on the coated plates after longer cultures, which indicated a higher proliferative capacity in response to COL1 and FN. RT-PCR, Western Blotting and mineralization assays did not reveal significant differences between the coated and noncoated surfaces in relation to osteogenic differential potential. Our data suggested that the surface coating of COL1 and FN were able to promote cellular proliferation and the osteogenic differentiation tendency of DPCs was also observed in vitro.

Bladder Cancer Chemosensitivity is Affected by Paraoxonase-2 Expression.

The goal of the current study was to identify potential roles of paraoxonase-2 in bladder carcinogenesis. T24 bladder cancer cells were transfected with plasmids inducing paraoxonase-2 silencing or overexpression. Upon the selection of clones stably down- or upregulating paraoxonase-2, cell proliferation, migration, and the production of reactive oxygen species were evaluated, before and after treatment with cisplatin and gemcitabine, used alone or in combination. The activity levels of both caspase-3 and caspase-8 were also analyzed. shRNA-mediated gene silencing and the overexpression of paraoxonase-2 revealed that the enzyme was able to promote both the proliferation and migration of T24 cells. Moreover, the knockdown of paraoxonase-2 was significantly associated with a reduced cell viability of T24 cells treated with chemotherapeutic drugs and led to both an increase of reactive oxygen species production and caspase-3 and caspase-8 activation. Conversely, under treatment with anti-neoplastic compounds, a higher proliferative capacity was found in T24 cells overexpressing paraoxonase-2 compared with controls. In addition, upon enzyme upregulation, both the production of reactive oxygen species and activation of caspase-3 and caspase-8 were reduced. Although further analyses will be required to fully understand the involvement of paraoxonase-2 in bladder tumorigenesis and in mechanisms leading to the development of chemoresistance, the data reported in this study seem to demonstrate that the enzyme could exert a great impact on tumor progression and susceptibility to chemotherapy, thus suggesting paraoxonase-2 as a novel and interesting molecular target for effective bladder cancer treatment.

Advances in generating liver cells from pluripotent stem cells as a tool for modeling liver diseases.

Developing robust in vitro models of the liver is essential for studying the pathogenesis of liver diseases, hepatotoxicity testing, and regenerative medicine. Earlier studies were conducted using cell lines derived from hepatomas. Due to the inherent limitations of cell lines, researchers used primary human hepatocytes (PHHs), which are considered a gold standard for in vitro modeling of the liver. However, due to the high cost of PHHs and lack of donors, researchers have sought an alternative source for functional liver cells. Pluripotent stem cells (PSCs) emerged as a viable alternative due to their plasticity and high proliferative capacity. This review gives an overview of the major advances that have been achieved to develop protocols to generate liver cells such as hepatocytes, cholangiocytes, and Küpffer cells from PSCs. We also discuss their application in modeling the pathogenesis of liver diseases such as drug‐induced liver injury, acute liver failure, and hepatic steatosis.

AICAR and nicotinamide treatment synergistically augment the proliferation and attenuate senescence-associated changes in mesenchymal stromal cells

BackgroundMesenchymal stromal cell (MSC) stemness capacity diminishes over prolonged in vitro culture, which negatively affects their application in regenerative medicine. To slow down the senescence of MSCs, here, we have evaluated the in vitro effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, and nicotinamide (NAM), an activator of sirtuin1 (SIRT1).MethodsHuman adipose-derived MSCs were cultured to passage (P) 5. Subsequently, the cells were grown in either normal medium alone (control group), the medium supplemented with AICAR (1 mM) and NAM (5 mM), or in the presence of both for 5 weeks to P10. Cell proliferation, differentiation capacity, level of apoptosis and autophagy, morphological changes, total cellular reactive oxygen species (ROS), and activity of mTORC1 and AMPK were compared among different treatment groups.ResultsMSCs treated with AICAR, NAM, or both displayed an increase in proliferation and osteogenic differentiation, which was augmented in the group receiving both. Treatment with AICAR or NAM led to decreased expression of β-galactosidase, reduced accumulation of dysfunctional lysosomes, and characteristic morphologic features of young MSCs. Furthermore, while NAM administration could significantly reduce the total cellular ROS in aged MSCs, AICAR treatment did not. Moreover, AICAR-treated cells possess a high proliferation capacity; however, they also show the highest level of cellular apoptosis. The observed effects of AICAR and NAM were in light of the attenuated mTORC1 activity and increased AMPK activity and autophagy.ConclusionsSelective inhibition of mTORC1 by AICAR and NAM boosts autophagy, retains MSCs’ self-renewal and multi-lineage differentiation capacity, and postpones senescence-associated changes after prolonged in vitro culture. Additionally, co-administration of AICAR and NAM shows an additive or probably a synergistic effect on cellular senescence.

In situ forming oxidized salecan/gelatin injectable hydrogels for vancomycin delivery and 3D cell culture

Antibiotics are widely used in clinical medicine. As an important member, vancomycin often plays an irreplaceable role in some serious infections, but its use still lacks suitable carriers and effective formulations. In order to find a vancomycin carrier with potential for clinical application, a new class of oxidized salecan/gelatin based injectable hydrogels are constructed through dynamic covalent Schiff base reaction. The sodium periodate oxidized salecan (OS) precursor was synthesized, and then the gelatin/oxidized salecan (GS) hydrogels are formed by blending gelatin and OS buffer solutions without any additives under physiological condition. The chemical structure, as well as internal morphologies, mechanical properties, In vitro enzymatic degradation profile of hydrogels are investigated with proton nuclear magnetic resonance (1H NMR), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), compression test and rheological experiments. The resulted hydrogels exhibit excellent antibacterial ability and variable characteristics. Moreover, the hydrogels display ideal drug release kinetics and mechanisms, and are applied successfully to the controlled release of vancomycin. Importantly, benefitting from the excellent biocompatibility and the reversibly crosslinked networks, GS hydrogels can function as suitable three dimensional (3D) extracellular matrix for HeLa cells, leading to the encapsulated cells maintaining a high viability and proliferative capacity. Therefore, the injectable GS hydrogels demonstrated attractive properties for future application in pharmaceutics and tissue engineering.

Highly Efficient Differentiation of Human Pluripotent Stem Cells into Pancreatic Progenitors Co-expressing PDX1 and NKX6.1.

Diabetes is a complex metabolic disorder, with no available treatment. Islet transplantation is currently practiced beta cell replacement therapy option, however, with major limitations. Human pluripotent stem cells (hPSCs) can be used as a scalable source for generation of insulin-secreting cells as hPSCs have high proliferative capacity and can differentiate into any tissue type. In vitro stepwise protocols have been designed for differentiating hPSCs into pancreatic lineages that finally give rise to beta cells; however, these hPSC-derived beta cells are dissimilar to adult human beta cells in key aspects of gene expression and functionality. Alternatively, pancreatic progenitors, when transplanted in the body, have been shown to mature into functional insulin-secreting beta cells, capable of reversing hyperglycemia. These pancreatic progenitors require the co-expression of PDX1, a transcription factor (TF) regulating pancreatic development, and NKX6.1, another TF key for beta cell maturation and function, to produce glucose-responsive beta cells. Given the crucial role played by NKX6.1, we optimized an in vitro differentiation protocol to enhance the generation of pancreatic progenitors co-expressing PDX1 and NKX6.1 by modulating cell density, matrix availability, and cellular dissociation.

Od nukleisanih do ex vivo manipulisanih matičnih ćelija - osavremenjena biološka i klinička sinapsa

Hematopoietic stem cells (SCs) are responsible for the production and replacement (proliferation) of an extensive quantity of functionally competent blood cells (differentiation) during the entire life, while simultaneously maintaining the ability to reproduce themselves (self-renewal). A complex network of interactive substances and factors organize and protect the survival, maturation and multiplication of SCs. Hemobiological events in the bone marrow (BM) are synchronized and balanced by the extracellular matrix and microenvironment provided by stromal cells. These cells-including macrophages, fibroblasts, dendritic, endothelial and other cells-stimulate SCs by producing specific hematopoietic growth factors. Other cytokines secreted by stromal cells regulate the adhesion molecules positioned on SCs, allowing them to remain in the BM or migrate to an area where the respective cell type is needed. Thus, hematopoietic SCs could be defined as cells with high proliferative capacity and extensive potential to differentiate into all blood cells or some somatic cell types (SC plasticity)-such as cardiomyocytes, myocytes, osteocytes, chondrocytes, hepatocytes, and even endothelial cells. Recent increasing clinical use of cell-mediated therapeutic approaches has resulted in increased needs for SCs, but in superior operating procedures during their ex vivo manipulations. The aim of cell harvestings is to obtain a higher SC yield and improved viability or clonogenicity. The goal of optimized cryoinvestigation protocols is to get a minimized cell damages (cryoinjury). Despite the fact that different SC collection protocols and cell freezing practice are already in routine use, a lot of questions related to the optimal SC ex vivo manipulations are still unresolved. This review summarizes fundamental knowledge and methodological approaches, and recapitulates data enabling progress on constantly evolving research frontiers in the SC area. The studies (including also our investigations) that evaluated the efficiency and safety of SC-treatment (transplants and regenerative medicine) will be also concisely presented.

Scalable Manufacturing of Human Hematopoietic Stem/Progenitor Cells Exploiting a Co-culture Platform with Mesenchymal Stromal Cells.

In the context of hematopoietic cell transplantation, hematopoietic stem/progenitor cells (HSPC) from the umbilical cord blood (UCB) present several advantages compared to adult sources including higher proliferative capacity, abundant availability and ease of collection, non-risk and painless harvesting procedure, and lower risk of graft-versus-host disease. However, the therapeutic utility of UCB HSPC has been limited to pediatric patients due to the low cell frequency per unit of UCB. The development of efficient and cost-effective strategies to generate large numbers of functional UCB HSPC ex vivo would boost all current and future medical uses of these cells. Herein, we describe a scalable serum-free co-culture system for the expansion of UCB-derived CD34+-enriched cells using microcarrier-immobilized human bone marrow-derived mesenchymal stromal cells as feeder cells.

Safety and efficacy of stem cell therapy for treatment of neural damage in patients with multiple sclerosis.

Multiple sclerosis (MS) is a multifocal inflammatory disease that involves the central nervous system and associated with limbs paralysis and serious problems in sensation, limbs, visual and sphincter. This disease is a result of autoimmune mechanism in which autoantibodies target the self-myelin antigens and cause demyelination. Because of the myelin dysfunction, MS is clinically identified with neurological disabilities. Furthermore, it can be entered into the progressive phase because of irreversible neurodegeneration and axons damage. Unfortunately, there is no effective therapeutic method for this disease and current medications have been focused on amelioration of symptoms and chronic inflammation. Although current immunotherapies ameliorate the reactivity of autoimmune anti-myelin and MS relapse rate, there is no approved method for improvement of the disease progression and repairing of the damaged myelin. Therefore, finding an appropriate clinical treatment for improvement of neurological damages in MS patients is essential. Mesenchymal stem cells (MSCs) are multipotent cells with high proliferative and self-renewal capacities, as well as immunomodulatory and neuroregenerative effects. Bone marrow and adipose tissues derived MSCs have been considered for the treatment of different diseases because not only they can be easily isolated from these tissues, but also a patient can be served as a donor for himself without the risk of rejection. More importantly, autologous MSCs carry a safer pattern without the risk of malignant transformation. Here, we will discuss the effectiveness of MSCs therapy for MS patients by reviewing of clinical trials.

Efficacy of third-party chimeric antigen receptor modified peripheral blood natural killer cells for adoptive cell therapy of B-cell precursor acute lymphoblastic leukemia.

We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19+ cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective "off-the-shelf" immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.

Establishment and characterization of proliferating primary cultures of equine epidermal keratinocytes

Skin-derived tissue cultures are a useful model to study molecular mechanisms of skin renewal and pathogenesis of dermal diseases. Horses often suffer from skin diseases, skin trauma and problems with proper wound healing, which could be improved by in vitro grown keratinocyte grafts. Herein we describe establishment and characterization of equine skin-derived primary cell cultures, using enzymatic and explant methods. The established cell lines of primary equine keratinocytes (peK) maintained high proliferative capacity for over five passages and expressed different epithelial/keratinocyte-specific markers. Characterization of the primary culture was performed in parallel with localization studies of the markers in the skin histological sections, using commercially available antibodies. Relative expression of typical differentiation stage-specific markers was determined in the established cell lines, using RT-qPCR. Basal (proliferating) keratinocytes were the predominant cell type in the established cell lines, but low expression of post-mitotic keratinocyte markers was also detected. Differences in marker expression were observed neither between the peK originating from two different animals nor between the peK established with two different methods (enzymatically or by explanting). The described methods in combination with the suggested characterization and differentiation markers are suitable for establishment of proliferating peK and evaluation of their differentiation status.

Sex-dependent VEGF expression underlies variations in human pluripotent stem cell to endothelial progenitor differentiation

Human pluripotent stem cells (hPSCs) offer tremendous promise in tissue engineering and cell-based therapies because of their unique combination of two properties: pluripotency and a high proliferative capacity. To realize this potential, development of efficient hPSC differentiation protocols is required. In this work, sex-based differences are identified in a GSK3 inhibitor based endothelial progenitor differentiation protocol. While male hPSCs efficiently differentiate into CD34 + CD31+ endothelial progenitors upon GSK3 inhibition, female hPSCs showed limited differentiation capacity using this protocol. Using VE-cadherin-GFP knockin reporter cells, female cells showed significantly increased differentiation efficiency when treated with VEGF during the second stage of endothelial progenitor differentiation. Interestingly, male cells showed no significant change in differentiation efficiency with VEGF treatment, but did show augmented early activation of VE-cadherin expression. A sex-based difference in endogenous expression of VEGF was identified that is likely the underlying cause of discrepancies in sex-dependent differentiation efficiency. These findings highlight the importance of sex differences in progenitor biology and the development of new stem cell differentiation protocols.

Evaluation of Ki-67 Expression in Oral Submucous Fibrosis and Its Correlation with Clinical and Histopathological Features.

Objectives:Oral submucous fibrosis (OSMF) is a potentially malignant disorder. Although it shows atrophic epithelium, it has a high proliferative capacity. Therefore, this study correlates the Ki-67. (The name “ki” is derived from the city of origin [Kiel, Germany] expression with functional grading and epithelial thickness in OSMF).Methods:The study group comprised of thirty patients of OSMF, divided randomly into Group A, Group B, Group C, and Group D as per mouth opening (functional staging). Five participants without OSMF formed the negative control group. The positive control group comprised of five patients of diagnosed cases of squamous cell carcinoma. All the sections of biopsy were subjected for hematoxylin and eosin and immunohistochemistry staining and observed for expression of Ki-67. Epithelial thickness was evaluated using image analysis software of Leica research microscope. Images were analyzed by three independent observers who were blindfolded. All the findings were tabulated and statistically analyzed.Results:In the present study, as the functional staging increased, the Ki-67 expression also increased. Ki-67 expression was highest in severe functional staging/severely decreased mouth opening (100.78) and is least in mild functional staging/mild decreased mouth opening (10.39). However, there was no significant correlation between epithelial thickness and functional staging/mouth opening (P > 0.05).Conclusion:A decrease in functional staging (mouth opening) showed a greater expression of Ki-67, and there was no significant correlation between functional staging and epithelial thickness.

Mesenchymal stem cells ascarrier of the therapeutic agent in the gene therapy of blood disorders.

Nonhematopoietic stem cells as a delivery platform of therapeutic useful genes have attracted widespread attention in recent years, owing to gained a long lifespan, easy separation, high proliferation, and high transfection capacity. Mesenchymal stem/stromal cells (MSCs) are the choice of the cells for gene and cell therapy due to high self-renewal capacity, high migration rate to the site of the tumor, and with immune suppressive and anti-inflammatory properties. Hence, it has a high potential of safety genetic modification of MSCs for antitumor gene expression and has paved the way for the clinical application of these cells to target the therapy of cancers and other diseases. The aim of gene therapy is targeted treatment of cancers and diseases through recovery, change, or enhancement cell performance to the sustained secretion of useful therapeutic proteins and induction expression of the functional gene in intended tissue. Recent developments in the vectors designing leading to the increase and durability of expression and improvement of the safety of the vectors that overcome a lot of problems, such as durability of expression and the host immune response. Nowadays, gene therapy approach is used by MSCs as a delivery vehicle in the preclinical and the clinical trials for the secretion of erythropoietin, recombinant antibodies, coagulation factors, cytokines, as well as angiogenic inhibitors in many blood disorders like anemia, hemophilia, and malignancies. In this study, we critically discuss the status of gene therapy by MSCs as a delivery vehicle for the treatment of blood disorders. Finally, the results of clinical trial studies are assessed, highlighting promising advantages of this emerging technology in the clinical setting.

Comparative Analysis of Wharton’s Jelly Mesenchymal Stem Cell (WJ-MSCs) Isolated Using Explant and Enzymatic Methods

Wharton’s Jelly is one of the best sources for mesenchymal stem cells. Human Wharton’s Jelly Mesenchymal Stem Cells (hWJ-MSCs) have high proliferation, multi-lineage differentiation potential, and do not produce any teratogen or carcinogen. These characteristics make hWJ-MSCs become suitable for regenerative medicine. Some methods were developed to isolate hWJ-MSCs from umbilical cord, such as explant method and enzymatic method. This study aims to characterize hWJ-MSCs which are isolated by two different methods, explant attachment method and enzymatic method. hWJ-MSCs isolation was performed through explant method and enzymatic method using trypsin, hyaluronidase and collagenase type 1 with certain ratio of concentration. Isolated hWJ-MSCs was characterized using flow cytometer to detect the expression of CD44, CD90, CD105, CD73 and negative lineage. MSCs differentiation assay was performed to analyze adipogenic, chondrogenic and osteogenic cells lineage. We successfully isolated hWJ-MSCs from umbilical cord through enzymatic and explant methods. Immunophenotyping assay through flow cytometry analysis showed high purity of WJ-MSCs. The isolated hWJ-MSCs from both methods showed positive expression of CD44, CD90, CD105, and CD73. The isolated hWJ-MSCs exhibited capacity to differentiate into adipocyte, chondrocyte, and osteocyte cells. hWJ-MSCs isolated through explant and enzymatic method have high proliferation capacity and be able to differentiate into three different lineage cells. Both methods explant attachment and enzymatic methods are efficiently produced hWJ-MSCs.

Combination of Human Amniotic Fluid Derived-Mesenchymal Stem Cells and Nano-hydroxyapatite Scaffold Enhances Bone Regeneration.

BACKGROUND:Human amniotic fluid-derived stem cells (hAF-MSCs) have a high proliferative capacity and osteogenic differentiation potential in vitro. The combination of hAF-MSCs with three-dimensional (3D) scaffold has a promising therapeutic potential in bone tissue engineering and regenerative medicine. Selection of an appropriate scaffold material has a crucial role in a cell supporting and osteoinductivity to induce new bone formation in vivo.AIM:This study aimed to investigate and evaluate the osteogenic potential of the 2nd-trimester hAF-MSCs in combination with the 3D scaffold, 30% Nano-hydroxyapatite chitosan, as a therapeutic application for bone healing in the induced tibia defect in the rabbit.SUBJECT AND METHODS:hAF-MSCs proliferation and culture expansion was done in vitro, and osteogenic differentiation characterisation was performed by Alizarin Red staining after 14 & 28 days. Expression of the surface markers of hAF-MSCs was assessed using Flow Cytometer with the following fluorescein-labelled antibodies: CD34-PE, CD73-APC, CD90-FITC, and HLA-DR-FITC. Ten rabbits were used as an animal model with an induced defect in the tibia to evaluate the therapeutic potential of osteogenic differentiation of hAF-MSCs seeded on 3D scaffold, 30% Nano-hydroxyapatite chitosan. The osteogenic differentiated hAF-MSCs/scaffold composite system applied and fitted in the defect region and non-seeded scaffold was used as control. The histopathological investigation was performed at 2, 3, & 4 weeks post-transplantation and scanning electron microscope (SEM) was assessed at 2 & 4 weeks post-transplantation to evaluate the bone healing potential in the rabbit tibia defect.RESULTS:Culture and expansion of 2nd-trimester hAF-MSCs presented high proliferative and osteogenic potential in vitro. Histopathological examination for the transplanted hAF-MSCs seeded on the 3D scaffold, 30% Nano-hydroxyapatite chitosan, demonstrated new bone formation in the defect site at 2 & 3 weeks post-transplantation as compared to the control (non-seeded scaffold). Interestingly, the scaffold accelerated the osteogenic differentiation of AF-MSCs and showed complete bone healing of the defect site as compared to the control (non-seeded scaffold) at 4 weeks post-transplantation. Furthermore, the SEM analysis confirmed these findings.CONCLUSION:The combination of the 2nd-trimester hAF-MSCs and 3D scaffold, 30% Nano-hydroxyapatite chitosan, have a therapeutic perspective for large bone defect and could be used effectively in bone tissue engineering and regenerative medicine.