Future Cell Therapy Research Articles

Comparative Evaluation of Differentiation Potential of Menstrual Blood- Versus Bone Marrow- Derived Stem Cells into Hepatocyte-Like Cells

Menstrual blood has been introduced as an easily accessible and refreshing stem cell source with no ethical consideration. Although recent works have shown that menstrual blood stem cells (MenSCs) possess multi lineage differentiation capacity, their efficiency of hepatic differentiation in comparison to other stem cell resources has not been addressed so far. The aim of this study was to investigate hepatic differentiation capacity of MenSCs compared to bone marrow-derived stem cells (BMSCs) under protocols developed by different concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM) in combination with other components in serum supplemented or serum-free culture media. Such comparison was made after assessment of immunophenotye, trans-differentiation potential, immunogenicity and tumorigeicity of these cell types. The differential expression of mature hepatocyte markers such as albumin (ALB), cytokeratin 18 (CK-18), tyrosine aminotransferase and cholesterol 7 alpha-hydroxylase activities (CYP7A1) at both mRNA and protein levels in differentiating MenSCs was significantly higher in upper concentration of HGF and OSM (P1) compared to lower concentration of these factors (P2). Moreover, omission of serum during differentiation process (P3) caused typical improvement in functions assigned to hepatocytes in differentiated MenSCs. While up-regulation level of ALB and CYP7A1 was higher in differentiated MenSCs compared to driven BMSCs, expression level of CK-18, detected level of produced ALB and glycogen accumulation were lower or not significantly different. Therefore, based on the overall comparable hepatic differentiation ability of MenSCs with BMSCs, and also accessibility, refreshing nature and lack of ethical issues of MenSCs, these cells could be suggested as an apt and safe alternative to BMSCs for future stem cell therapy of chronic liver diseases.

Amniotic fluid stem cells for minimally invasive prenatal cell therapy

Abstract Amniotic fluid stem cells can be expanded without feeder layers and can differentiate into mesenchymal and hematopoietic lineages. Long-term engraftment has been difficult to achieve after prenatal stem cell transplantation, mainly because of allogeneic rejection. Autologous cells can be obtained from amniotic fluid with minimal risk. This review aims to define the potential of human amniotic fluid stem cells as an autologous stem cell source for minimal invasive prenatal cell or cell-based gene therapy. We conclude that amniotic fluid-derived stem cells are an important source of autologous cells that may have prenatal therapeutic value in cell or cell-based gene therapy in the near future.

Successful Induction of Induced Pluripotent Stem Cells (iPSC's) from Human Granulosa Cells (hGC's)

It has been recently discovered that somatic cells can be reprogrammed into iPSC's by transgenic expression of a key transcription factors (OCT4, SOX2, KLF4, and cMYC). These iPSC's mimic the pluripotent state observed for human embryonic stem cells (ESC's) and have the advantage that human embryos are not needed, thus circumventing potential ethical concerns. In addition, this reprogramming technology has the potential to generate patient-specific iPSC's for future cell therapy or disease modeling to develop novel therapies for infertile patients.

Sequential introduction and dosage balance of defined transcription factors affect reprogramming efficiency from pancreatic duct cells into insulin-producing cells

While the exogenous expression of a combination of transcription factors have been shown to induce the conversion of non-β cells into insulin-producing cells, the reprogramming efficiency remains still low. In order to develop an in vitro screening system for an optimized reprogramming protocol, we generated the reporter cell line mPac-MIP-RFP in which the reprogramming efficiency can be quantified with red fluorescent protein expressed under the control of the insulin promoter. Analysis with mPac-MIP-RFP cells sequentially infected with adenoviruses expressing Pdx1, Neurog3, and Mafa revealed that expression of Pdx1 prior to Neurog3 or Mafa augments the reprogramming efficiency. Next, infection with a polycistronic adenoviral vector expressing Pdx1, Neurog3 and Mafa significantly increased the expression level of insulin compared with the simultaneous infection of three adenoviruses carrying each transcription factor, although excessive expression of Mafa together with the polycistronic vector dramatically inhibited the reprogramming into insulin-producing cells. Thus, in vitro screening with the mPac-MIP-RFP reporter cell line demonstrated that the timing and dosage of gene delivery with defined transcription factors influence the reprogramming efficiency. Further investigation should optimize the reprogramming conditions for the future cell therapy of diabetes.

Improved Efficiency of Microsurgical Enucleated Tripronuclear Zygotes Development and Embryonic Stem Cell Derivation by Supplementing Epidermal Growth Factor, Brain-Derived Neurotrophic Factor, and Insulin-Like Growth Factor-1

Human embryonic stem cells (hESCs) hold great promise for future clinical cell therapies because of their unique potential to differentiate into all human cell types. However, the destruction of normal fertilized embryos and the derivation of hESCs for research has resulted in polarized ethical debates, with most of the controversy centered on embryo destruction. Therefore, due to less ethical controversy surrounding them, abnormal fertilized zygotes that are usually discarded are a potential feasible resource for the derivation of hESCs. Microsurgery on human polyspermic zygotes can contribute to the derivation of hESCs, but the efficiency is much lower. Here, we reported a culture system to enhance the developmental competence of such microsurgical human polyspermic zygotes by EGF-BDNF-IGF-1 combination, which eventually resulted in the increased derivation efficiency of hESCs from them. We found that the developmental efficiency of microsurgical enucleated tripronuclear (3PN) embryos cultured with the EGF-BDNF-IGF-1 combination was significantly increased compared with the control group. More importantly, when the microsurgical enucleated 3PN embryos were cultured in medium supplemented with EGF-BDNF-IGF-1, the frequency ratio of chromosome abnormality was reduced. Our present study will facilitate the development of hESC line derivation in subsequent studies and also provide an additional choice for infertile couples.

Review: The future of cell therapies and brain repair: Parkinson's disease leads the way

During the past 40 years brain tissue grafting techniques have been used both to study fundamental neurobiological questions and to treat neurological diseases. Motor symptoms of Parkinson's disease are largely due to degeneration of midbrain dopamine neurones. Because the nigrostriatal pathology is relatively focused anatomically, Parkinson's disease is considered the ideal candidate for brain repair by neural grafting and dopamine neurone transplantation for it has led the way in the neural transplantation research field. In this mini-review, we briefly highlight four important areas of development. First, we describe marked functional benefits up to 18 years after transplantation surgery in patients with Parkinson's disease. This is proof-of-principle that, using optimal techniques and patient selection, grafted dopamine neurones can work in humans and the duration of the benefit exceeds placebo effects associated with surgery. Second, we describe that eventually protein aggregates containing α-synuclein, identical to Lewy bodies, develop inside foetal dopamine neurones transplanted to patients with Parkinson's disease. This gives clues about pathogenetic mechanisms operating in Parkinson's disease, and also raises the question whether neural graft function will eventually decline as the result of the disease process. Third, we describe new emerging sources of transplantable dopamine neurones derived from pluripotent stem cells or reprogrammed adult somatic cells. Fourth, we highlight an important European Union-funded multicentre clinical trial involving transplantation of foetal dopamine neurones in Parkinson's disease. We describe the design of this ongoing trial and how it can impact on the overall future of cell therapy in Parkinson's disease.

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Purinergic receptors and nucleotide processing ectoenzymes: Their roles in regulating mesenchymal stem cell functions.

Human mesenchymal stem cells (MSCs) are a rare population of non-hematopoietic stem cells with multilineage potential, originally identified in the bone marrow. Due to the lack of a single specific marker, MSCs can be recognized and isolated by a series of features such as plastic adherence, a panel of surface markers, the clonogenic and the differentiation abilities. The recognized role of MSCs in the regulation of hemopoiesis, in cell-degeneration protection and in the homeostasis of mesodermal tissues through their differentiation properties, justifies the current interest in identifying the biochemical signals produced by MSCs and their active crosstalk in tissue environments. Only recently have extracellular nucleotides (eNTPs) and their metabolites been included among the molecular signals produced by MSCs. These molecules are active on both ionotropic and metabotropic receptors present in most cell types. MSCs possess a significant display of these receptors and of nucleotide processing ectoenzymes on their plasma membrane. Thus, from their niche, MSCs give a significant contribution to the complex signaling network of eNTPs and its derivatives. Recent studies have demonstrated the multifaceted aspects of eNTP metabolism and their signal transduction in MSCs and revealed important roles in specifying differentiation lineages and modulating MSC physiology and communication with other cells. This review discusses the roles of eNTPs, their receptors and ectoenzymes, and the relevance of the signaling network and MSC functions, and also focuses on the importance of this emerging area of interest for future MSC-based cell therapies.

Abstract 18338: Biomaterial Delivery Vehicles Improve Acute Retention of Cells in the Infarcted Heart

Introduction and Hypothesis: Cell delivery to the infarcted heart has emerged as a promising therapy, but is limited by very low acute retention and engraftment of cells. The hypothesis was that acute retention can be improved with a biomaterial carrier, and the study compared several. Methods: Cells were quantified 24 hours post-implantation in a rat myocardial infarct model (Fig. 1a) in five groups (n=8 per group); saline injection (current clinical standard), two injectable gels (alginate and chitosan) and two epicardial patches (alginate and collagen). For injectable groups 60ul of saline or gel containing 400,000 human mesenchymal stem cells was injected intramyocardially in the infarct border zone. 400,000 cells were seeded on alginate or collagen patches, and implanted on the epicardial surface at infarct border zone. At 24 hours, retained cells were quantified with Xenogen imaging system. Hearts were perfused and sectioned to stain for retained cells. Results: All biomaterials significantly improved retention compared to a saline control, with 4.1 and 2.6-fold increases for chitosan and alginate injectables, and 14.2 and 19.3-fold increases achieved with collagen and alginate patches, respectively (Fig. 1b-c). Immunohistochemical analysis qualitatively confirmed these findings (Fig. 1d). Encapsulated/seeded cell survival was assessed in hypoxia/ischemia to further compare biomaterials. Conclusion: Injectable gels and epicardial patches were demonstrated to improve acute retention of cells when compared to a saline control. Injectable gels enable immediate myocardial delivery while epicardial patches facilitate superior retention and could potentially sustain cell delivery over extended periods. These biomaterial approaches should be considered for future cell therapy applications. Figure 1: a) Procedural steps b-c) Cell retention after 24 hours d) Ventricle wall showing DAPI and implanted cells with GFP antibody (green) for each group

English

Introduction Chronic obstructive pulmonary disease is a degenerative, life-threatening lung disease defined by a persistent poor airflow. Pulmonary vascular endothelial dysfunction is a characteristic pathological finding of chronic obstructive pulmonary disease. Endothelial progenitor cells are a population of adult stem cells mobilised from the bone marrow into the circulation, with the function of maintaining vascular homeostasis. Decreasing number of endothelial progenitor cells has been established as a prognostic risk factor associated with endothelial dysfunction and high cardiovascular risk. In chronic obstructive pulmonary disease, few studies to date have investigated the number of endothelial progenitor cells with conflicting results. Quantification of endothelial progenitor cells is affected by high individual variability. Age, gender, smoking habit, cardiovascular comorbidities, different cell populations examined, different methodologies used or the sample size of the study, all are variables that can influence endothelial progenitor cell numbers. This review tries to comprehend why these discrepancies in endothelial progenitor cell numbers occur and sets strategies to encourage greater confidence in the reproducibility of the results, a basis for future stem cell therapy studies. Conclusion Greater confidence in the reproducibility of the quantification of EPCs taking into account all possible variables is necessary before using them as the basis for detailed mechanistic investigations and stem cell therapy. Introduction Chronic obstructive pulmonary disease Chronic obstructive pulmonary disease (COPD) is a degenerative, lifethreatening lung disease, affecting an estimated 329 million people and accounting for 5% of all deaths globally per year1. Mortality is expected to increase due to a rise in smoking rates and an aging population in many countries. The World Health Organization predicts that COPD will become the third leading cause of death worldwide by 20301. Unfortunately, COPD is not curable and cannot be reversed. Drug treatment can improve symptoms and minimise further damage, but does not alter the underlying progression of this disease. Thus, COPD is a major public health problem with high prevalence, increasing incidence and elevated associated socioeconomic costs. Post-bronchodilator spirometry is required for the diagnosis and assessment of severity of COPD2. Is defined by a persistent poor airflow (FEV1/FVC < 0.70), which results from an inflammatory process affecting the airways and lung parenchyma usually in the form of emphysema2. Chronic and acute inflammation induced by the inhalation of noxious particles, mainly cigarette smoking (CS), is the primary pathogenesis of COPD3. Genetic factors and aging effects are also involved in the disease progression. Vascular change and angiogenesis is an integral part of the pulmonary inflammatory response. The lung is exposed to external challenges such as inhaled particles, toxic gases or infections every day; the defence against these external injuries depends on the immune system and the efficiency of the system to remove and replace apoptotic injured cells4. CS not only causes inflammation and direct injury to lung tissue but also inhibits compensatory angiogenesis and thus impairs repair and adaptive mechanisms in the lung. The aim of this review was to discuss endothelial progenitor cells (EPCs) in COPD. Discussion COPD and endothelial dysfunction Pulmonary vascular endothelial dysfunction is a characteristic pathological finding of COPD. Studies have shown alterations in the vessel structure and an abnormal functional associated with an atypical control of endothelial cellular growth and resistance to apoptosis in the endothelium of patients suffering from COPD5,6. Growing evidence suggests that the endothelial cell damage and dysfunction found in pulmonary vessels of these patients is an initial and important triggering factor that promotes pulmonary vasculature remodelling and, hence, pulmonary hypertension in COPD7. It is been recognised that patients with COPD showed an endothelial impairment in both pulmonary and systemic circulation at early stages of the disease4,8. In addition, scanning electron microscopy of the endothelial surface * Corresponding author Email: tura@clinic.ub.es Department of Pulmonary Medicine, Hospital Clinic-Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), University of Barcelona Centro de Investigacion Biomedica en Red de Enfermedades Respiratorias, Spain 08036 Barcelona, Spain.

Implementation of Cardovascular Cell Therapy Network trials: challenges, innovation and lessons learned from experience in the CCTRN

The cardiovascular cell therapy network was developed by the National Heart, Lung and Blood Institute to design and conduct clinical trials to advance the field of cardiovascular (CV) cell-based therapy. The Cardiovascular Cell Therapy Network successfully completed three clinical trials involving approximately 300 subjects across five centers and six satellites. Although the concept of a network within clinical trials research is not new, the knowledge gained in the implementation of such large-scale trials, particularly in novel therapeutic areas such as cell therapy is not often detailed in the literature. The purpose of this communication is to summarize key factors in achieving network goals and share the knowledge gained to promote success in future cardiovascular disease cell therapy trials and networks.

Alginate encapsulation parameters influence the differentiation of microencapsulated embryonic stem cell aggregates.

Pluripotent embryonic stem cells (ESCs) have tremendous potential as tools for regenerative medicine and drug discovery, yet the lack of processes to manufacture viable and homogenous cell populations of sufficient numbers limits the clinical translation of current and future cell therapies. Microencapsulation of ESCs within microbeads can shield cells from hydrodynamic shear forces found in bioreactor environments while allowing for sufficient diffusion of nutrients and oxygen through the encapsulation material. Despite initial studies examining alginate microbeads as a platform for stem cell expansion and directed differentiation, the impact of alginate encapsulation parameters on stem cell phenotype has not been thoroughly investigated. Therefore, the objective of this study was to systematically examine the effects of varying alginate compositions on microencapsulated ESC expansion and phenotype. Pre-formed aggregates of murine ESCs were encapsulated in alginate microbeads composed of a high or low ratio of guluronic to mannuronic acid residues (High G and High M, respectively), with and without a poly-L-lysine (PLL) coating, thereby providing four distinct alginate bead compositions for analysis. Encapsulation in all alginate compositions was found to delay differentiation, with encapsulation within High G alginate yielding the least differentiated cell population. The addition of a PLL coating to the High G alginate prevented cell escape from beads for up to 14 days. Furthermore, encapsulation within High M alginate promoted differentiation toward a primitive endoderm phenotype. Taken together, the findings of this study suggest that distinct ESC expansion capacities and differentiation trajectories emerge depending on the alginate composition employed, indicating that encapsulation material physical properties can be used to control stem cell fate.

Cardiac Hegemony of Senescence

Cardiac senescence and age-related disease development have gained general attention and recognition in the past decades due to increased accessibility and quality of health care. The advancement in global civilization is complementary to concerns regarding population aging and development of chronic degenerative diseases. Cardiac degeneration has been rigorously studied. The molecular mechanisms of cardiac senescence are on multiple cellular levels and hold a multilayer complexity level, thereby hampering development of unambiguous treatment protocols. In particular, the synergistic exchange of the senescence phenotype through a senescence secretome between myocytes and stem cells appears complicated and is of great future therapeutic value. The current review article will highlight hallmarks of senescence, cardiac myocyte and stem cell senescence, and the mutual exchange of senescent secretome. Future cardiac cell therapy approaches require a comprehensive understanding of myocardial senescence to improve therapeutic efficiency as well as efficacy.

Effect of a single autologous cord blood infusion on beta-cell and immune function in children with new onset type 1 diabetes: a non-randomized, controlled trial

The application of autologous cord blood in children with type 1 diabetes has been found to be safe, but not to preserve beta-cell function in a previous study, which, however, had not included a control group. To compare the changes of metabolic and immune function over time between cord blood infused children and natural controls. Seven children with newly diagnosed type 1 diabetes underwent a single autologous cord blood infusion and 10 children were enrolled as natural controls in a non-randomized, controlled, open label intervention trial. Primary analyses were performed 1 year following cord blood infusion. Cases and controls were compared regarding metabolic [area under the curve (AUC) and peak C-peptide, insulin use, and HbA1c] and immune outcome (islet autoantibody titer and T-cell response), adjusted for age, gender, diabetes duration, and baseline levels. There were no significant adverse events related to the infusion. Metabolic and immune outcomes were not significantly different at 12 months follow-up between infused children and controls (e.g., adjusted p = 0.244 for AUC C-peptide, adjusted p = 0.820 for insulin use, adjusted p = 0.772 for peripheral regulatory T cells). Six-month change of AUC C-peptide correlated significantly with the number of infused CD34+ cells (r = 0.931, p = 0.002). An autologous cord blood infusion does not change the natural course of metabolic and immune parameters after disease onset. However, the content of CD34+ cells in the stored blood sample might offer potential for improvement of future cell therapies.

Pancreatic Differentiation of Human Dental Pulp CD117 + stem cells

Adult stem cells cannot proliferate to produce enough cells for human transplantation with keeping stem cell characteristics shown in the primary culture. We established a novel culture protocol using human dental pulp stem cells (DPSCs) that can produce quantities sufficient for human transplantation. The present study assessed differentiation of DPSCs toward a pancreatic lineage in serum-free conditions, which is essential for safe transplantation. CD117⁺ stem cells were separated from human exfoliated deciduous teeth (stem cells from human exfoliated deciduous teeth; SHED) and adult DPSCs. The cells were characterized with real-time reverse-transcription PCR for a panel of embryonal lineage markers. 82 out of 84 markers were expressed in different levels in SHED or DPSCs. After pancreatic differentiation in vitro, we found expression of pancreatic-specific endocrine markers insulin, glucagon, somatostatin and pancreatic polypeptide, and exocrine marker amylase-2a in both cultures. We also found reprogramming in both cell cultures mimicking the embryonal stages of development of the pancreas. Transcription factors PDX1, HHEX, MNX1, NEUROG3, PAX4, PAX6 and NKX6-1, crucial markers for the pancreatic development, were all activated. Expression of these factors strongly implies that the cells differentiated toward a distinguished pancreatic lineage. Our results show that CD117⁺ SHED and DPSCs are capable of differentiation toward all functional endocrine and exocrine subsets of pancreatic cells in serum-free conditions. SHED and DPSCs may therefore have great potential for future cell therapy of pancreatic disorders.

The developmental origin of postnatal cardiac regeneration

Recent studies have shown that postnatal mammalian hearts undergo cardiomyocyte refreshment. However, evidence is lacking for the cell origin of these postnatal cardiomyogenesis. We first confirmed that myocardial injury triggered the expression of embryonic cardiogenesis genes at the external part of the heart. To determine whether cardiac lineage-specific progenitor cells exist in the postnatal mammalian heart and have cardiomyogenic potential, we created coronary artery ligation on the Nkx2.5 enhancer-eGFP transgenic mice. The number of Nkx2.5 enhancer-eGFP cells was increasing after myocardial injury and peaked at 2 weeks post injury. The activated eGFP+ cells expressed cardiac precursor markers. The eGFP signaling was not expressed in mature cardiomyocytes, hematopoietic cells or fibroblasts. These activated Nkx2.5 enhancer-eGFP cells would differentiate into striated cardiomyocytes in vitro. We further confirmed that cardiac progenitor cells contributed directly to postnatal cardiomyogenesis by lineage tracing using inducible Cre system. To trace the developmental origin of the activated Nkx2.5 enhancer-eGFP cells, we created different lineage-Cre/Nkx2.5 enhancer-eGFP/ ROSA26 reporter triple transgenic mice. The post-injury Nkx2.5 enhancer-eGFP+ cells originated from the embryonic epicardial cells, but did not originate from the pre-existing cardiomyocytes, endothelial cells, cardiac neural crest cells, and perinatal/ postnatal epicardial cells. Taken together, our data confirmed that cardiac-lineage progenitor cells, which originated from embryonic epicardium-derived cells, contributed to postnatal mammamian cardiomyogenesis. The discovery of cardiomyogenic cell population in the postnatal heart enables the future cell therapy for cardiac regeneration.

Transplantation of NSCs with OECs alleviates neuropathic pain associated with NGF downregulation in rats following spinal cord injury

Spinal cord injury (SCI) is a common and serious disease which often induces catastrophic consequence in patients. Part of them exhibit neuropathic pain which presents unique challenges to clinicians, and there is no effective approach for the treatment up to now. Neural stem cells (NSCs) transplantation, as a promising and an effective method, could be considered for the treatment of SCI, whereas a main problem is the low survival of NSCs in traumatic milieu in host spinal cords, and the effect of NSCs on sensory function remains elusive. In this study, we investigated the effect and underlying molecular mechanism of co-transplantation of NSCs with olfactory ensheathing cells (OECs) on sensory functional improvement. In the measurement of thermal and mechanical stimuli, NSCs grafts recovered sensory function in SCI rats, while OECs led to hyperalgesia, indicated by the tail flick latency (TFL) and paw withdraw latency (PWL) (p<0.05). Co-transplantation could promote NSCs survival, and reverses the hyperalgesia triggered by OECs. This was corresponding to a significant improvement in sensory function. Moreover, NGF expression was substantial downregulated in the spinal cord of co-transplantation rats. The present findings suggested that co-transplantation of NSCs with OECs could improve sensory function and the possible mechanism is involved in NGF downregulation in rats with SCI. This may give some new indications for the treatment of SCI in future clinic cell therapy trial.

Human conditionally immortalized neural stem cells improve locomotor function after spinal cord injury in the rat

IntroductionA growing number of studies have highlighted the potential of stem cell and more-differentiated neural cell transplantation as intriguing therapeutic approaches for neural repair after spinal cord injury (SCI).MethodsA conditionally immortalized neural stem cell line derived from human fetal spinal cord tissue (SPC-01) was used to treat a balloon-induced SCI. SPC-01 cells were implanted into the lesion 1 week after SCI. To determine the feasibility of tracking transplanted stem cells, a portion of the SPC-01 cells was labeled with poly-L-lysine-coated superparamagnetic iron-oxide nanoparticles, and the animals grafted with labeled cells underwent magnetic resonance imaging. Functional recovery was evaluated by using the BBB and plantar tests, and lesion morphology, endogenous axonal sprouting and graft survival, and differentiation were analyzed. Quantitative polymerase chain reaction (qPCR) was used to evaluate the effect of transplanted SPC-01 cells on endogenous regenerative processes.ResultsTransplanted animals displayed significant motor and sensory improvement 2 months after SCI, when the cells robustly survived in the lesion and partially filled the lesion cavity. qPCR revealed the increased expression of rat and human neurotrophin and motor neuron genes. The grafted cells were immunohistologically positive for glial fibrillary acidic protein (GFAP); however, we found 25% of the cells to be positive for Nkx6.1, an early motor neuron marker. Spared white matter and the robust sprouting of growth-associated protein 43 (GAP43)+ axons were found in the host tissue. Four months after SCI, the grafted cells matured into Islet2+ and choline acetyltransferase (ChAT)+ neurons, and the graft was grown through with endogenous neurons. Grafted cells labeled with poly-L-lysine-coated superparamagnetic nanoparticles before transplantation were detected in the lesion on T2-weighted images as hypointense spots that correlated with histologic staining for iron and the human mitochondrial marker MTCO2.ConclusionsThe transplantation of SPC-01 cells produced significant early functional improvement after SCI, suggesting an early neurotrophic action associated with long-term restoration of the host tissue, making the cells a promising candidate for future cell therapy in patients with SCI.

Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide a promising source for cell therapy and drug screening. Several high-yield protocols exist for hESC-CM production; however, methods to significantly advance hESC-CM maturation are still lacking. Building on our previous experience with mouse ESC-CMs, we investigated the effects of 3-dimensional (3D) tissue-engineered culture environment and cardiomyocyte purity on structural and functional maturation of hESC-CMs. 2D monolayer and 3D fibrin-based cardiac patch cultures were generated using dissociated cells from differentiated Hes2 embryoid bodies containing varying percentage (48–90%) of CD172a (SIRPA)-positive cardiomyocytes. hESC-CMs within the patch were aligned uniformly by locally controlling the direction of passive tension. Compared to hESC-CMs in age (2 weeks) and purity (48–65%) matched 2D monolayers, hESC-CMs in 3D patches exhibited significantly higher conduction velocities (CVs), longer sarcomeres (2.09 ± 0.02 vs. 1.77 ± 0.01 μm), and enhanced expression of genes involved in cardiac contractile function, including cTnT, αMHC, CASQ2 and SERCA2. The CVs in cardiac patches increased with cardiomyocyte purity, reaching 25.1 cm/s in patches constructed with 90% hESC-CMs. Maximum contractile force amplitudes and active stresses of cardiac patches averaged to 3.0 ± 1.1 mN and 11.8 ± 4.5 mN/mm2, respectively. Moreover, contractile force per input cardiomyocyte averaged to 5.7 ± 1.1 nN/cell and showed a negative correlation with hESC-CM purity. Finally, patches exhibited significant positive inotropy with isoproterenol administration (1.7 ± 0.3-fold force increase, EC50 = 95.1 nm). These results demonstrate highly advanced levels of hESC-CM maturation after 2 weeks of 3D cardiac patch culture and carry important implications for future drug development and cell therapy studies.