Human embryonic stem cell-derived mesenchymal progenitors—Potential in regenerative medicine

Tissue engineering and cell therapy require large-scale production of homogeneous populations of lineage-restricted progenitor cells that easily can be induced to differentiate into a specific tissue. We have developed straightforward protocols for the establishment of human embryonic stem (hES) cell-derived mesenchymal progenitor (hES-MP) cell lines. The reproducibility was proven by derivation of multiple hES-MP cell lines from 10 different hES cell lines. To illustrate clinical applicability, a xeno-free hES-MP cell line was also derived. None of the markers characteristic for undifferentiated hES cells were detected in the hES-MP cells. Instead, these cells were highly similar to mesenchymal stem cells with regard to morphology and expression of markers. The safety of hES-MP cells following transplantation was studied in severely combined immunodeficient (SCID) mice. The implanted hES-MP cells gave rise to homogeneous, well-differentiated tissues exclusively of mesenchymal origin and no teratoma formation was observed. These cells further have the potential to differentiate toward the osteogenic, adipogenic, and chondrogenic lineages in vitro. The possibility of easily and reproducibly generating highly expandable hES-MP cell lines from well-characterized hES cell lines with differentiation potential into several mesodermal tissues entails an enormous potential for the field of regenerative medicine.     

Bone marrow mesenchymal stem cell transplantation improves radiation-induced heart injury through DNA damage repair in rat model

Radiotherapy is an effective form of therapy for most thoracic malignant tumors. However, myocardial injury resulting from the high doses of radiation is a severe complication. Here we aimed to study the possibility of reducing radiation-induced myocardial injury with mesenchymal stem cell (MSC) transplantation. We used MSCs extracted from bone marrow (BMSCs) to transplant via the tail vein into a radiation-induced heart injury (RIHI) rat model. The rats were divided into six groups: a Sham group, an IRR (irradiation) group, and four IRR + BMSCs transplantation groups obtained at different time points. After irradiation, BMSC transplantation significantly enhanced the cardiac function in rats. By analyzing the expression of PPAR-α, PPAR-γ, TGF-β, IL-6, and IL-8, we found that BMSC transplantation alleviated radiation-induced myocardial fibrosis and decreased the inflammatory reaction. Furthermore, we found that expression of γ-H2AX, XRCC4, DNA ligase4, and TP53BP1, which are associated with DNA repair, was up-regulated, along with increased secretion of growth factors SDF-1, CXCR4, VEGF, and IGF in rat myocardium in the IRR + BMSCs transplantation groups compared with the IRR group. Thus, BMSC transplantation has the potential to improve RIHI via DNA repair and be a new therapeutic approach for patients with myocardial injury.     

对Beitz大鼠后肢穿刺伤疼痛模型进行改良的实验研究

目的:对Beitz大鼠后肢穿刺伤疼痛模型进行改良.方法:雄性SD大鼠35只,200±20g.将其随机分为假手术组(C)5只,Beitz模型组(BM),改良Beitz模型组(IM)各15只.按照Beitz大鼠后肢穿刺伤模型要求制作,改良Beitz模型组左小腿外侧距膝关节1cm处做纵行切口长1.0cm,并垂直刺入肌肉达对侧皮肤.其余步骤同于Beitz模型.造模后分别测定各实验组动物的继发机械痛阈、继发热痛阈和脊髓背角Fos阳性蛋白的积分光密度(IOD),并进行比较.结果:改良后的动物模型也可反应出急性疼痛的演化过程,较原有模型敏感度提高.结论:改良后的Beitz模型组可以模拟急性疼痛的发生发展过程,而且适合对局部镇痛方法的镇痛效果进行评价.     

Human mesenchymal stromal cell transplantation modulates neuroinflammatory milieu in a mouse model of amyotrophic lateral sclerosis

Background aimsMesenchymal stromal cells (MSCs), after intraparenchymal, intrathecal and endovenous administration, have been previously tested for cell therapy in amyotrophic lateral sclerosis in the SOD1 (superoxide dismutase 1) mouse. However, every administration route has specific pros and cons.MethodsWe administrated human MSCs (hMSCs) in the cisterna lumbaris, which is easily accessible and could be used in outpatient surgery, in the SOD1 G93A mouse, at the earliest onset of symptoms. Control animals received saline injections. Motor behavior was checked starting from 2 months of age until the mice were killed. Animals were killed 2 weeks after transplantation; lumbar motoneurons were stereologically counted, astrocytes and microglia were analyzed and quantified after immunohistochemistry and cytokine expression was assayed by means of real-time polymerase chain reaction.ResultsWe provide evidence that this route of administration can exert strongly positive effects. Motoneuron death and motor decay were delayed, astrogliosis was reduced and microglial activation was modulated. In addition, hMSC transplantation prevented the downregulation of the anti-inflammatory interleukin-10, as well as that of vascular endothelial growth factor observed in saline-treated transgenic mice compared with wild type, and resulted in a dramatic increase in the expression of the anti-inflammatory interleukin-13.ConclusionsOur results suggest that hMSCs, when intracisternally administered, can exert their paracrine potential, influencing the inflammatory response of the host.     

Functional recovery after transplantation of bone marrow-derived human mesenchymal stromal cells in a rat model of spinal cord injury

Background aimsSpinal cord injury (SCI) is a medically untreatable condition for which stem cells have created hope. Pre-clinical and clinical studies have established that these cells are safe for transplantation. The dose dependency, survivability, route of administration, cell migration to injury site and effect on sensory and motor behavior in an SCI-induced paraplegic model were studied.MethodsA spinal cord contusion injury model was established in rats. Bone marrow (BM) mesenchymal stromal cells (MSC) were tagged to facilitate tracing in vivo. Two different doses (2 and 5 million cells/kg body weight) and two different routes of infusion (site of injury and lumbar puncture) were tested during and after the spinal shock period. The animals were tested post-transplantation for locomotor capacity, motor control, sensory reflex, posture and body position. Stem cell migration was observed 1 month post-transplantation in spinal cord sections.ResultsThe overall results demonstrated that transplantation of BM MSC significantly improved the locomotor and sensory behavior score in the experimental group compared with the sham control group, and these results were dose dependent. All the infused stem cells could be visualized at the site of injury and none was visualized at the injected site. This indicated that the cells had survived in vivo, were probably chemoattracted and had migrated to the lesion site.ConclusionsMSC transplanted with a lumbar puncture method migrate to the site of injury and are the most suitable for SCI healing. These cells demonstrate a dose-dependent effect and promote functional recovery when injected during or after the spinal shock period.     

Lipocalin 2 enhances mesenchymal stem cell-based cell therapy in acute kidney injury rat model

Acute kidney injury (AKI) is one of the most common health-threatening diseases in the world. There is still no effective medical treatment for AKI. Recently, Mesenchymal stem cell (MSC)-based therapy has been proposed for treatment of AKI. However, the microenvironment of damaged kidney tissue is not favorable for survival of MSCs which would be used for therapeutic intervention. In this study, we genetically manipulated MSCs to up-regulate lipocalin-2 (Lcn2) and investigated whether the engineered MSCs (MSC-Lcn2) could improve cisplatin-induced AKI in a rat model. Our results revealed that up-regulation of Lcn2 in MSCs efficiently enhanced renal function. MSC Lcn2 up-regulates expression of HGF, IGF, FGF and VEGF growth factors. In addition, they reduced molecular biomarkers of kidney injury such as KIM-1 and Cystatin C, while increased the markers of proximal tubular epithelium such as AQP-1 and CK18 following cisplatin-induced AKI. Overall, here we over-expressed Lcn2, a well-known cytoprotective factor against acute ischemic renal injury, in MSCs. This not only potentiated beneficial roles of MSCs for cell therapy purposes but also suggested a new modality for treatment of AKI.     

Human embryonic mesenchymal stem cell-derived conditioned medium rescues kidney function in rats with established chronic kidney disease

Chronic kidney disease (CKD) is a major health care problem, affecting more than 35% of the elderly population worldwide. New interventions to slow or prevent disease progression are urgently needed. Beneficial effects of mesenchymal stem cells (MSC) have been described, however it is unclear whether the MSCs themselves or their secretome is required. We hypothesized that MSC-derived conditioned medium (CM) reduces progression of CKD and studied functional and structural effects in a rat model of established CKD. CKD was induced by 5/6 nephrectomy (SNX) combined with L-NNA and 6% NaCl diet in Lewis rats. Six weeks after SNX, CKD rats received either 50 μg CM or 50 μg non-CM (NCM) twice daily intravenously for four consecutive days. Six weeks after treatment CM administration was functionally effective: glomerular filtration rate (inulin clearance) and effective renal plasma flow (PAH clearance) were significantly higher in CM vs. NCM-treatment. Systolic blood pressure was lower in CM compared to NCM. Proteinuria tended to be lower after CM. Tubular and glomerular damage were reduced and more glomerular endothelial cells were found after CM. DNA damage repair was increased after CM. MSC-CM derived exosomes, tested in the same experimental setting, showed no protective effect on the kidney. In a rat model of established CKD, we demonstrated that administration of MSC-CM has a long-lasting therapeutic rescue function shown by decreased progression of CKD and reduced hypertension and glomerular injury.     

Enrichment of vascular endothelial growth factor secreting mesenchymal stromal cells enhances therapeutic angiogenesis in a mouse model of hind limb ischemia

Critical limb ischemia, a severe manifestation of peripheral artery disease, is emerging as a major concern in aging societies worldwide. Notably, cell-based gene therapy to induce angiogenesis in ischemic tissue has been investigated as treatment. Despite many studies demonstrating the efficacy of this approach, better therapies are required to prevent serious sequelae such as claudication, amputation and other cardiovascular events. We have now established a simplified method to enhance the effects of therapeutic transgenes by selecting for and transplanting only transduced cells. Herein, mesenchymal stromal cells were transfected to co-express vascular endothelial growth factor as angiogenic factor and enhanced green fluorescent protein as marker. Transfected cells were then collected using flow cytometry based on green fluorescence and transplanted into ischemic hind limbs in mice. Compared with unsorted or untransfected cells, purified cells significantly improved blood perfusion within 21days, suggesting that transplanting only cells that overexpress vascular endothelial growth factor enhances therapeutic angiogenesis. Importantly, this approach may prove to be useful in cell-based gene therapy against a wide spectrum of diseases, simply by replacing the gene to be delivered or the cell to be transplanted.     

The effect of a nitroxide antioxidant on ischemia-reperfusion injury in the rat in vivo hind limb model

Microsurgical procedures such as free tissue transfer or replantations of amputated digits involve an obligatory ischemic period leading to regional tissue oedema, rhabdomyolysis, systemic acidosis, hypercalcemia and multiple organ dysfunction syndrome reflecting ischemia-reperfusion (I/R) injury. Since nitroxide stable radicals act as antioxidants their potential protective effects were tested. Anaesthetized Sabra rats were subjected to regional ischemia of the hind limb for 2 h using a tourniquet. Upon reperfusion rats were injected with 4-OH-2,2,6,6-tetramethylpiperidine-1-oxyl (TPL). Systemic I/R-induced damage was assessed by sampling blood for differential count, lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) serum levels. Regional injury was evaluated by analysing excised muscle samples for oedema (tissue water content) and inflammatory infiltrate (number of cell nuclei in histomorphometric analysis). I/R-induced changes of biomarkers reflecting systemic damage peaked about 8 h following the start of reperfusion and fully disappeared as the biomarkers relaxed to their pre-ischemic values after 24 h. TPL facilitated the recovery of some of these parameters and partially affected release of cellular CPK and LDH. The parameters of I/R-induced regional tissue injury did not demonstrate any recovery and were not inhibited by TPL.     

The effect of Trimetazidine and Diazoxide on immunomodulatory activity of human embryonic stem cell-derived mesenchymal stem cell secretome

Comprehensive proteome profiling of the factors secreted by mesenchymal stem cells (MSCs), referred to as secretome, revealed that it consists of cytokines, chemokines, growth factors, extracellular matrix proteins, and components of regeneration, vascularization, and hematopoiesis pathways. Harnessing this MSC secretome for therapeutic applications requires the optimization of production of secretary molecules. A variety of preconditioning methods have been introduced, which subject cells to stimulatory molecules to create the preferred response and stimulate persistent effects. Pharmacological preconditioning uses small molecules and drugs to increase survival of MSCs after transplantation or prolong release of effective secretary factors such as cytokines that improve immune system responses. In this study, we investigated the effect of secretome of human embryonic-derived mesenchymal stem cells (hESC-MSCs) preconditioned with Trimetazidine (TMZ) and Diazoxide (DZ) on immunomodulatory efficiency of these cells in LPS-induced peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from human peripheral blood and treated with concentrated hESC-MSC-derived conditioned medium and then, the secreted levels of IL-10, TNFα and IL-1β were assessed by ELISA after induction with LPS. The results showed that TMZ and DZ-conditioned medium significantly enhanced immunomodulatory potential of hESC-MSCs by increasing the secretion of IL-10, TNFα and IL-1β from LPS- induced PBMCs. We also found that hESC-MSCs did not secrete mentioned cytokines prior to or after the preconditioning with TMZ and DZ. In conclusion, our results implied that TMZ and DZ can be used to promote the immunomodulatory effects of hESC-MSC secretome. It is obvious that for applying of these findings in clinical demands, the potency of different pre-conditioned MSCs secretome on immune response needs to be more clarified.     

Human umbilical cord mesenchymal stem cell transplantation restores damaged ovaries

Ovarian injury because of chemotherapy can decrease the levels of sexual hormones and potentia generandi of patients, thereby greatly reducing quality of life. The goal of this study was to investigate which transplantation method for human umbilical cord mesenchymal stem cells (HUMSCs) can recover ovarian function that has been damaged by chemotherapy. A rat model of ovarian injury was established using an intraperitoneal injection of cyclophosphamide. Membrane‐labelled HUMSCs were subsequently injected directly into ovary tissue or tail vein. The distribution of fluorescently labelled HUMSCs, estrous cycle, sexual hormone levels, and potentia generandi of treated and control rats were then examined. HUMSCs injected into the ovary only distributed to the ovary and uterus, while HUMSCs injected via tail vein were detected in the ovary, uterus, kidney, liver and lung. The estrous cycle, levels of sex hormones and potentia generandi of the treated rats were also recovered to a certain degree. Moreover, in some transplanted rats, fertility was restored and their offspring developed normally. While ovary injection could recover ovarian function faster, both methods produced similar results in the later stages of observation. Therefore, our results suggest that transplantation of HUMSCs by tail vein injection represents a minimally invasive and effective treatment method for ovarian injury.     

The effect of a nitroxide antioxidant on ischemia-reperfusion injury in the rat in vivo hind limb model

Microsurgical procedures such as free tissue transfer or replantations of amputated digits involve an obligatory ischemic period leading to regional tissue oedema, rhabdomyolysis, systemic acidosis, hypercalcemia and multiple organ dysfunction syndrome reflecting ischemia-reperfusion (I/R) injury. Since nitroxide stable radicals act as antioxidants their potential protective effects were tested. Anaesthetized Sabra rats were subjected to regional ischemia of the hind limb for 2 h using a tourniquet. Upon reperfusion rats were injected with 4-OH-2,2,6,6-tetramethylpiperidine-1-oxyl (TPL). Systemic I/R-induced damage was assessed by sampling blood for differential count, lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) serum levels. Regional injury was evaluated by analysing excised muscle samples for oedema (tissue water content) and inflammatory infiltrate (number of cell nuclei in histomorphometric analysis). I/R-induced changes of biomarkers reflecting systemic damage peaked about 8 h following the start of reperfusion and fully disappeared as the biomarkers relaxed to their pre-ischemic values after 24 h. TPL facilitated the recovery of some of these parameters and partially affected release of cellular CPK and LDH. The parameters of I/R-induced regional tissue injury did not demonstrate any recovery and were not inhibited by TPL.     

Human embryonic stem cell-derived cardiomyocytes as an in vitro model to study cardiac insulin resistance

Patients with type 2 diabetes (T2D) and/or insulin resistance (IR) have an increased risk for the development of heart failure (HF). Evidence indicates that this increased risk is linked to an altered cardiac substrate preference of the insulin resistant heart, which shifts from a balanced utilization of glucose and long-chain fatty acids (FAs) towards an almost complete reliance on FAs as main fuel source. This shift leads to a loss of endosomal proton pump activity and increased cardiac fat accumulation, which eventually triggers cardiac dysfunction. In this review, we describe the advantages and disadvantages of currently used in vitro models to study the underlying mechanism of IR-induced HF and provide insight into a human in vitro model: human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Using functional metabolic assays we demonstrate that, similar to rodent studies, hESC-CMs subjected to 16 h of high palmitate (HP) treatment develop the main features of IR, i.e., decreased insulin-stimulated glucose and FA uptake, as well as loss of endosomal acidification and insulin signaling. Taken together, these data propose that HP-treated hESC-CMs are a promising in vitro model of lipid overload-induced IR for further research into the underlying mechanism of cardiac IR and for identifying new pharmacological agents and therapeutic strategies. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

Human umbilical cord mesenchymal stromal cells mitigate chemotherapy-associated tissue injury in a pre-clinical mouse model

Background aimsMesenchymal stromal cells (MSC) have been shown to be a promising candidate for tissue regeneration and cancer therapy. However, their therapeutic potential against chemotherapy-induced side-effects remains unclear.MethodsWe treated murine Lewis lung carcinoma (LLC) and xenograft human colon tumors with adriamycin (ADM) for 3 consecutive days followed by one intravenous (i.v.) injection of human umbilical cord (hUC) MSC for several cycles.ResultsMSC treatment mitigated ADM-induced cardiomyopathy, reduced the extent of ADM-induced apoptosis in intestinal crypts, suppressed body weight loss in mice treated with ADM and increased the survival rate of mice treated with a lethal dose of ADM. The examination of hematologic parameters indicated a moderate recovery in MSC-injected mice. Systemic administration of MSC did not increase the growth of murine LLC cells and human colon carcinoma in vivo while it strongly inhibited the lung metastases of LLC cells.ConclusionsWe evaluated the prophylactic and therapeutic action of hUC MSC on the chemotherapy agent ADM-induced side-effects in two different tumor models. Our observations suggest that MSC can be used as auxiliary means in chemotherapy for certain tumor types.     

Adherent self-renewable human embryonic stem cell-derived neural stem cell line: functional engraftment in experimental stroke model

Background

Human embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.

Methods/Principal Findings

We isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.

Conclusions/Significance

The SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research.     

Human mesenchymal stem cell biology

This brief overview summarises the main characteristics of bone marrow mesenchymal stem cells and of adipose-derived stem cells: methods of obtention, phenotype, differentiation potential, hematopoiesis-supportive (stromal) capacity, and immunosuppressive properties. Two points are discussed in detail: 1) criteria for stemness: multipotency, self-renewal, plasticity, and 2) the repair mechanisms implicated in the different indications of cell therapy using these cells: reconstitution of the tissue functional compartment by repopulation consequent to proliferation and differentiation or reprogrammation, stromal effects by secretion of angiogenic, anti-apoptotic, anti-fibrogenic factors, molecules involved in the regulation of inflammation, etc.     

Human embryonic stem cell and embryonic germ cell lines

Undifferentiated human embryonic stem (ES) cells and embryonic germ (EG) cells can be cultured indefinitely and yet maintain the potential to form many or all of the differentiated cells in the body. Human ES and EG cells provide an exciting new model for understanding the differentiation and function of human tissue, offer new strategies for drug discovery and testing, and promise new therapies based on the transplantation of ES and EG cell-derived tissues.