Therapy For Degenerative Diseases Research Articles

Regulation of Immune Checkpoint Antigen CD276 (B7-H3) on Human Placenta-Derived Mesenchymal Stromal Cells in GMP-Compliant Cell Culture Media.

Therapies utilizing autologous mesenchymal cell delivery are being investigated as anti-inflammatory and regenerative treatments for a broad spectrum of age-related diseases, as well as various chronic and acute pathological conditions. Easily available allogeneic full-term human placenta mesenchymal stromal cells (pMSCs) were used as a potential pro-regenerative, cell-based therapy in degenerative diseases, which could be applied also to elderly individuals. To explore the potential of allogeneic pMSCs transplantation for pro-regenerative applications, such cells were isolated from five different term-placentas, obtained from the dissected maternal, endometrial (mpMSCs), and fetal chorion tissues (fpMSCs), respectively. The proliferation rate of the cells in the culture, as well as their shape, in vitro differentiation potential, and the expression of mesenchymal lineage and stem cell markers, were investigated. Moreover, we studied the expression of immune checkpoint antigen CD276 as a possible modulation of the rejection of transplanted non-HLA-matched homologous or even xeno-transplanted pMSCs. The expression of the cell surface markers was also explored in parallel in the cryosections of the relevant intact placenta tissue samples. The expansion of pMSCs in a clinical-grade medium complemented with 5% human platelet lysate and 5% human serum induced a significant expression of CD276 when compared to mpMSCs expanded in a commercial medium. We suggest that the expansion of mpMSCs, especially in a medium containing platelet lysate, elevated the expression of the immune-regulatory cell surface marker CD276. This may contribute to the immune tolerance towards allogeneic pMSC transplantations in clinical situations and even in xenogenic animal models of human diseases. The endurance of the injected comparably young human-term pMSCs may promote prolonged effects in clinical applications employing non-HLA-matched allogeneic cell therapy for various degenerative disorders, especially in aged adults.

Optimizing apothecary through medicinal plant cultivation and education of elderly posyandu cadres in Tegalwaras

As people age, the prevalence of degenerative diseases increases. These diseases can reduce the quality of life and often cause fatal effects, and this happens a lot in Tegalwaras hamlet. Treatment with modern medicine sometimes causes problems such as relatively expensive prices and dangerous side effects so alternatives such as herbal medicines are needed. This community service aims to increase the knowledge of Posyandu Lansia cadres in Tegalwaras about herbs as a therapy for degenerative diseases through counseling and planting medicinal plants. The method of implementation is the preparation stage, counseling, planting medicinal plants, monitoring and evaluation. The results of the program showed an increase in knowledge of 50% after the counseling was carried out.

Research Status and Prospect of Non-Viral Vectors Based on siRNA: A Review

Gene therapy has attracted much attention because of its unique mechanism of action, non-toxicity, and good tolerance, which can kill cancer cells without damaging healthy tissues. siRNA-based gene therapy can downregulate, enhance, or correct gene expression by introducing some nucleic acid into patient tissues. Routine treatment of hemophilia requires frequent intravenous injections of missing clotting protein. The high cost of combined therapy causes most patients to lack the best treatment resources. siRNA therapy has the potential of lasting treatment and even curing diseases. Compared with traditional surgery and chemotherapy, siRNA has fewer side effects and less damage to normal cells. The available therapies for degenerative diseases can only alleviate the symptoms of patients, while siRNA therapy drugs can upregulate gene expression, modify epigenetic changes, and stop the disease. In addition, siRNA also plays an important role in cardiovascular diseases, gastrointestinal diseases, and hepatitis B. However, free siRNA is easily degraded by nuclease and has a short half-life in the blood. Research has found that siRNA can be delivered to specific cells through appropriate vector selection and design to improve the therapeutic effect. The application of viral vectors is limited because of their high immunogenicity and low capacity, while non-viral vectors are widely used because of their low immunogenicity, low production cost, and high safety. This paper reviews the common non-viral vectors in recent years and introduces their advantages and disadvantages, as well as the latest application examples.

Cationic molecular probes based on aggregation-induced emission for fluorescent sensing and super-resolution imaging of insulin fibrosis

Amyloid fibrosis of protein is one of protein-related essential issues, which provides the critical information for the diagnosis and therapy of degenerative diseases. Insulin possesses the determined structure and relatively low molecular weight, which is often used as the main model protein to study amyloid fibrosis. Here, we have designed and synthesized three hydrophilic cationic aggregation-induced emission (AIE) probes with different aromatic ketone structures through geminal cross-coupling (GCC) reaction. Cationic AIE molecular probes can bind to insulin fibrils with negative charge at pH = 9.0 through electrostatic interaction, which constrains the intramolecular motion of probes and enhances the fluorescence. The detection limit of probes for insulin fibrils is as low as 0.64 nM. The random and sparse lighting of cationic molecular probes can be realized to enable the super-resolution imaging through the reversible electrostatic association-dissociation interaction between the negative insulin fibrils and positive molecular probes. The fine structure of the fibrils is observed, and full-width at half maximum (FWHM) for assessing the resolution can reach 35 nm, which is of great significance for the development of high-performance super-resolution imaging probes based on aggregation-induced emission and their optical nanoscale visualization applications in the exploration of amyloid fibrillation and related diseases.

Targeted Polypeptide–Microtubule Aggregation with Cucurbit[8]uril for Enhanced Cell Apoptosis

AbstractTunable protein assemblies not only hold a dominant position in vital biological events but are also a significant theme in supramolecular chemistry. Herein, we demonstrated that the intertubular aggregation of microtubules (MTs) could be efficiently regulated by a synergistic polypeptide–tubulin interaction and host–guest complexation. The benzylimidazolium‐modified antimitotic peptide (BP) could recognize the MTs and concurrently form stable inclusion complexes with avirulent cucurbit[7]uril (CB[7]) and cucurbit[8]uril (CB[8]) in different binding stoichiometries. The self‐assembling morphology of MTs was converted from fibrous to nanoparticulate aggregates via extensive BP⊂CB[8] cross‐linkage, leading to significant cell apoptosis and tumor ablation in vivo. The targeted (BP⊂CB[8])@MT ternary assembly provides a facile supramolecular method to enhance the protein–protein interactions, which may be developed as a therapy for degenerative diseases, such as cancer.

Targeted Polypeptide-Microtubule Aggregation with Cucurbit[8]uril for Enhanced Cell Apoptosis.

Tunable protein assemblies not only hold a dominant position in vital biological events but are also a significant theme in supramolecular chemistry. Herein, we demonstrated that the intertubular aggregation of microtubules (MTs) could be efficiently regulated by a synergistic polypeptide-tubulin interaction and host-guest complexation. The benzylimidazolium-modified antimitotic peptide (BP) could recognize the MTs and concurrently form stable inclusion complexes with avirulent cucurbit[7]uril (CB[7]) and cucurbit[8]uril (CB[8]) in different binding stoichiometries. The self-assembling morphology of MTs was converted from fibrous to nanoparticulate aggregates via extensive BP⊂CB[8] cross-linkage, leading to significant cell apoptosis and tumor ablation in vivo. The targeted (BP⊂CB[8])@MT ternary assembly provides a facile supramolecular method to enhance the protein-protein interactions, which may be developed as a therapy for degenerative diseases, such as cancer.

Protein disulfide isomerase as a prosurvival factor in cell therapy for muscular and vascular diseases

BackgroundCell therapy for degenerative diseases aims at rescuing tissue damage by delivery of precursor cells. Thus far, this strategy has been mostly unsuccessful due to massive loss of donor cells shortly after transplantation. Several strategies have been applied to increase transplanted cell survival but only with limited success. The endoplasmic reticulum (ER) is an organelle involved in protein folding, calcium homeostasis, and lipid biosynthesis. Protein disulfide isomerase (PDI) is a molecular chaperone induced and activated by ER stress. PDI is induced by hypoxia in neuronal, cardiac, and endothelial cells, supporting increased cell survival to hypoxic stress and protection from apoptosis in response to ischemia.MethodsWe achieved ex vivo PDI gene transfer into luciferase-expressing myoblasts and endothelial cells. We assessed cell engraftment upon intramuscular transplantation into a mouse model of Duchenne muscular dystrophy (mdx mouse) and into a mouse model of ischemic disease.ResultsWe observed that loss of full-length dystrophin expression in mdx mice muscle leads to an increase of PDI expression, possibly in response to augmented ER protein folding load. Moreover, we determined that overexpression of PDI confers a survival advantage for muscle cells in vitro and in vivo to human myoblasts injected into murine dystrophic muscle and to endothelial cells administered upon hindlimb ischemia damage, improving the therapeutic outcome of the cell therapy treatment.ConclusionsCollectively, these results suggest that overexpression of PDI may protect transplanted cells from hypoxia and other possibly occurring ER stresses, and consequently enhance their regenerative properties.

Science for education: a new model of translational research applied to education

A great advance in the last transition of centuries has been the consolidation of the concept of translational research, applied with success in Health and Engineering in practically all countries of medium/high GDP. Intriguingly, this has not occurred with Education. It is yet not perceived that Science can already understand how people learn, which are the mechanisms that accelerate learning and teaching, and how this would impact on the economy and the social progress of nations. It is also not perceived that innovations can be validated with populational studies to rationalize and scale novel teaching initiatives, nor which socioemotional competences should future citizens possess to work in companies more and more automatized and informatized. Perhaps because of this omission, the progress of Brazilian educational indicators has been so modest. In Health, public policies not only invest in material improvements (sanitation, hospital attendance, nutritional coverture, etc), but also on Science and Innovation capable of creating new options in the international scenario (therapies for degenerative diseases, vaccines for infectious diseases, etc). Differently, on Education investment has focused exclusively on material improvements (more schools, better salaries for teachers, etc), necessary but insufficient to accelerate growth of our indicators at faster and more competitive rates. This scenario opens to us a window of opportunity to create a new Science policy aiming at Education. To give concreteness to this possibility, the proposal on discussion is that the new initiatives of support and funding by public and private agencies should have Science for Education as its structurant axis.

Therapeutic relevance of ozone therapy in degenerative diseases: Focus on diabetes and spinal pain.

Ozone, one of the most important air pollutants, is a triatomic molecule containing three atoms of oxygen that results in an unstable form due to its mesomeric structure. It has been well-known that ozone has potent ability to oxidize organic compounds and can induce respiratory irritation. Although ozone has deleterious effects, many therapeutic effects have also been suggested. Since last few decades, the therapeutic potential of ozone has gained much attention through its strong capacity to induce controlled and moderated oxidative stress when administered in precise therapeutic doses. A plethora of scientific evidence showed that the activation of hypoxia inducible factor-1α (HIF-1a), nuclear factor of activated T-cells (NFAT), nuclear factor-erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE), and activated protein-1 (AP-1) pathways are the main molecular mechanisms underlying the therapeutic effects of ozone therapy. Activation of these molecular pathways leads to up-regulation of endogenous antioxidant systems, activation of immune functions as well as suppression of inflammatory processes, which is important for correcting oxidative stress in diabetes and spinal pain. The present study intended to review critically the available scientific evidence concerning the beneficial properties of ozone therapy for treatment of diabetic complications and spinal pain. It finds benefit for integrating the therapy with ozone into pharmacological procedures, instead of a substitutive or additional option to therapy.

The influence of rAAV2‐mediated SOX2 delivery into neonatal and adult human RPE cells; a comparative study

Cell replacement is a promising therapy for degenerative diseases like age-related macular degeneration (AMD). Since the human retina lacks regeneration capacity, much attention has been directed toward persuading for cells that can differentiate into retinal neurons. In this report, we have investigated reprogramming of the human RPE cells and concerned the effect of donor age on the cellular fate as a critical determinant in reprogramming competence. We evaluated the effect of SOX2 over-expression in human neonatal and adult RPE cells in cultures. The coding region of human SOX2 gene was cloned into adeno-associated virus (AAV2) and primary culture of human neonatal/adult RPE cells were infected by recombinant virus. De-differentiation of RPE to neural/retinal progenitor cells was investigated by quantitative real-time PCR and ICC for neural/retinal progenitor cells' markers. Gene expression analysis showed 80-fold and 12-fold over-expression for SOX2 gene in infected neonatal and adult hRPE cells, respectively. The fold of increase for Nestin in neonatal and adult hRPE cells was 3.8-fold and 2.5-fold, respectively. PAX6 expression was increased threefold and 2.5-fold in neonatal/adult treated cultures. Howbeit, we could not detect rhodopsin, and CHX10 expression in neonatal hRPE cultures and expression of rhodopsin in adult hRPE cells. Results showed SOX2 induced human neonatal/adult RPE cells to de-differentiate toward retinal progenitor cells. However, the increased number of PAX6, CHX10, Thy1, and rhodopsin positive cells in adult hRPE treated cultures clearly indicated the considerable generation of neuro-retinal terminally differentiated cells.

Role of the Cell Cycle Re-Initiation in DNA Damage Response of Post-Mitotic Cells and Its Implication in the Pathogenesis of Neurodegenerative Diseases.

Neurodegenerative diseases are often associated with both normal and premature aging. Resumption of the cell cycle by neurons induced by DNA damage may lead to their apoptosis, which contributes to the degeneration of neuronal tissue. Cell cycle and DNA replication proteins are frequently found in patients with neurodegenerative diseases. Oxidative stress, which is considered to play an important role in aging and pathogenesis of many neurodegenerative diseases, can induce DNA damage and stimulate cell cycle re-entry by neuronal cells. DNA damage activates ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), breast cancer 1 (BRCA1), E2F transcription factor 1 (E2F1), and other proteins that regulate the cell cycle, DNA damage repair, and apoptosis. Because the E2F complexes associate with histone-modifying enzymes, histone modifications, including histone acetylation and methylation, are required for cell cycle re-entry and may play a regulatory role in DNA repair or apoptosis. Aberrant cell cycle regulation has been shown to play a role in age-related macular degeneration (AMD) in which retinal cells are affected and in inclusion body myositis, which is characterized by muscle cell dysfunction. There is also evidence to suggest that cytostatic chemotherapy could decrease dementia in Alzheimer's disease and multiple myeloma, supporting the use of cell cycle inhibitors in the therapy of degenerative diseases.

Microenvironmental Interaction Between Hypoxia and Endothelial Cells Controls the Migration Ability of Placenta-Derived Mesenchymal Stem Cells via α4 Integrin and Rho Signaling.

Mesenchymal stem cells (MSCs) are a powerful source for cell therapy in degenerative diseases. The migration ability of MSCs is an important factor that enhances the therapeutic effect of the cells when they are transplanted into target tissues or organs. Hypoxia and the endothelial barrier, which are representative migration microenvironmental factors, are known to be regulated by the integrin-mediated pathway in several cancers. However, their regulatory mechanisms in MSCs remain unclear. Here, the objectives of the study were to compare the expression of markers related to integrin-mediated signaling in placenta-derived MSCs (PDMSCs) dependent on hypoxia and co-cultured with human umbilical vein endothelial cells (HUVECs) and to evaluate their correlations between migration ability and microenvironmetal factors including hypoxia and endothelial cells. The migration abilities of PDMSCs exposed to hypoxic conditions were significantly increased compared with normal fibroblasts (WI-38) and control (P < 0.05). Interestingly, decreased integrin α4 in PDMSCs under hypoxia induce to increase migration abilities of PDMSCs. Also, Rho family-related markers were significantly increased in PDMSCs under hypoxic conditions compared with normoxia (P < 0.05). Furthermore, the migration ability of PDMSCs was decreased by Rho kinase inhibitor treatment (Y-27632) and co-culturing with HUVECs in an ex vivo system. ROCK activity was increased by inhibiting integrin α4 with HUVECs and hypoxia compared with the absence of HUVECs and under normoxia. The findings suggest microenvironment event by hypoxia and the interaction with endothelial cells may be useful as a regulator of MSC migration and provide insight into the migratory mechanism of MSCs in stem cell-based therapy.

Near-IR absorbing quantum dots might be usable for growth factor-based differentiation of stem cells

Abstract For stem cell therapy of degenerative diseases, it is necessary to differentiate stem cells into the specific lineage. There are several growth factors which have been used for differentiation of stem cells. Some growth factors can dose-dependently induce differentiation of stem cells so that the increase of growth factor concentration results in production of the higher level of differentiated cells. However, due to the toxicity of some differentiation factors (e.g. retinoic acid), the lower dose of growth factors for the specific lineage differentiation of stem cells is desirable. This paper suggests a new approach in the field of controlled growth factor delivery system using semiconductor nanocrystals; known as quantum dots (QDs). This system contains polymeric microencapsulated growth factor which is conjugated to near infrared (NIR) absorbing QDs. The control release of growth factors from microcapsules in the culture plates can be achieved by irradiation. To modulate growth factor release in response to stem cells needs for differentiation, the intensity and period of irradiation will be controlled. Our hypothesis is based on the fact that QDs can absorb NIR energy and by excitation of electrons and then vibrational relaxation of them become heated when they were irradiated and then release growth factors. We believe that controlled growth factors delivery through the suggested system is an effective method to reduce the amount of growth factors required for differentiation of stem cells.

Evaluation of analgesic effectiveness of infrared radiation and interference currents in degenerative diseases

AbstractBackground: The aim of this research is to evaluate analgesic effectiveness of infrared radiation and interference currents in degenerative diseases of joints. On the grounds of current practical and theoretical experience, the following hypothesis was formed: Application of interference currents and infrared radiation constitutes effective analgesic therapy in degenerative diseases, and in the case of the applied treatment, its effectiveness is long-term.Material/Methods: Tests were conducted on a group consisting of 32 women and men in the age range of 65-87 years of age suffering from a degenerative disease of the knee joint. The patients were applied a series of 10 treatments with application of the Sollux lamp for 15 minutes and interference currents of 50-100 HZ frequency for 5 minutes and 90- 100 Hz for 10 minutes during treatment. Evaluation of the efficiency of therapy was checked by means of the VAS scale and the Laitinen scale.Results: The results indicate an efficient analgesic effect according to the VAS scale immediately after treatment p = 0.002 and a month after treatment p = 0.000 as well as according to the Laitinen scale immediately after treatment p = 0.004 and a month after treatment p = 0.004.Conclusions: Application of infrared radiation and interference currents indicates an analgesic effect. These treatments may be alternative or supportive to pharmacological treatment in the case of degenerative changes in people who are over 65 years of age. Results of the research suggest continuation of further research on the effectiveness of the above-mentioned therapy and the length of preserving analgesic results in people with degenerative diseases of joints

Scavenging of hydroxyl radical by resveratrol and related natural stilbenes after hydrogen peroxide attack on DNA

Resveratrol (3,5,4′-trihydroxystilbene) is of interest due to its role in prevention and therapy of degenerative diseases as cancer and aging. However, depending on its concentration and cell type studied, resveratrol activity appears conflicting. It exerts antioxidant action, as a scavenger of free radicals and as promoter of antioxidant enzyme activity, but resveratrol acts also as a pro-oxidant. Here we present experimental and theoretical studies for resveratrol and two methoxy-derivatives found in plants, pterostilbene and 3,5,4′-trimethoxystilbene. We show that both methoxy-derivatives induce less DNA damage than resveratrol. The protective effects of the three molecules against oxidative DNA damage induced by hydrogen peroxide treatment were analyzed on mammalian cells in vitro. Our data show for the first time that methoxylated derivatives of resveratrol are very efficient in reducing DNA damage: using the same concentration of the three molecules we obtain a relative reduction of 85.5% (pterostilbene), 43.7% (trimethoxystilbene) and 21.1% (resveratrol). Analysis of the crystal structures of pterostilbene and 3,5,4′-trimethoxystilbene, compared to resveratrol, show fewer intermolecular interactions and a lack of planarity, due to packing forces, which is confirmed by density functional theory (DFT) calculations. We also describe the results of DFT calculations (including water solvent effects) in which the three stilbene species scavenge the hydroxyl radical (associated with the H2O2 insult).

Estradiol Synthesis and Release in Cultured Female Rat Bone Marrow Stem Cells

Bone marrow stem cells (BMSCs) have the capacity to differentiate into mature cell types of multiple tissues. Thus, they represent an alternative source for organ-specific cell replacement therapy in degenerative diseases. In this study, we demonstrated that female rat BMSCs could differentiate into steroidogenic cells with the capacity for de novo synthesis of Estradiol-17β (E2) under high glucose culture conditions with or without retinoic acid (RA). The cultured BMSCs could express the mRNA and protein for P450arom, the enzyme responsible for estrogen biosynthesis. Moreover, radioimmunoassay revealed that BMSCs cultured in the present culture system produced and secreted significant amounts of testosterone, androstenedione, and E2. In addition, RA promoted E2 secretion but did not affect the levels of androgen. These results indicate that BMSCs can synthesize and release E2 and may contribute to autologous transplantation therapy for estrogen deficiency.

Intracranial Stem Cell-Based Transplantation: Reconsidering the Ethics of Phase 1 Clinical Trials in Light of Irreversible Interventions in the Brain

Stem-cell-based transplantation is the subject of early clinical investigations as a possible therapy for degenerative diseases of the brain. Given the novelty of the strategy, the potential for direct benefit to research subjects is at best very uncertain. Yet conducting these trials for the sole or main purpose of generating safety data is ethically disputable because the risks and burdens of intracranial cell transplantation are such that they cannot be offset solely by the value of the generalizable knowledge expected to be gained. The typical rationale for delaying the initial assessment of efficacy to the second phase of clinical trials, that is, the need to first define a safe dose range, is not relevant to cell-based brain transplantation trials because stem cells are proliferative cells and transplants are irreversible. Given the intervention's high risks and burdens, and the probable involvement of subject populations unable to consent in a large proportion of future trials, there may be an ethical duty to make the assessment of clinically relevant efficacy a primary objective of phase 1 trials. This would entail maximizing the value of preclinical efficacy studies, incorporating primary efficacy endpoints in the protocols, and selecting subject populations with an open therapeutic window. In addition, initial safety and efficacy data for a specific product should be first collected in consenting adults whenever possible. If such conditions were met, aspirational therapeutic goals would attenuate the ethically contentious nature of these trials, even in the absence of a clear therapeutic warrant.

Targeted Cell Fusion Facilitates Stable Heterokaryon Generation In Vitro and In Vivo

Induced cell fusion has enabled several important discoveries, including the phenomenon of nuclear reprogramming and may yet be applied as a novel therapy for degenerative diseases. However, existing fusogens lack the efficiency required to enable investigation of the epigenetic modifications underlying nuclear reprogramming and the specificity required for clinical application. Here we present a chimeric measles hemagglutinin, Hα7, which specifically and efficiently mediates the fusion of diverse cell types with skeletal muscle both in vitro and in vivo. When compared directly to polyethylene glycol, Hα7 consistently generated a substantial increase in heterokaryon yield and exhibited insignificant levels of toxicity. Moreover, this increased fusion efficiency enabled detection of chromatin modifications associated with nuclear reprogramming following Hα7-mediated fusion of human fibroblasts and mouse myotubes. Finally, Hα7 was also capable of increasing the contribution of transplanted fibroblasts to skeletal muscle repair in vivo, suggesting that this strategy could be used for therapeutic gene delivery.

Embryonic Stem Cell-Derived Hematopoietic Stem Cells: Challenges in Development, Differentiation, and Immunogenicity

Embryonic stem cells (ESC) can potentially be manipulated in vitro to differentiate into cells and tissues of all three germ layers. This pluripotent feature is being exploited to use ESC-derived tissues as therapies for degenerative diseases and replacement of damaged organs. Although their potential is great, the promise of ESC-derived therapies will be unfulfilled unless several challenges are overcome. For example, inefficient production of ESC-derived tissues before transplantation, inability of ESC-derived tissues to integrate well into the adult microenvironments due to developmental stage incompatibility, or active immune rejection of the ESC-derived graft are all potential challenges to successful ESC-derived therapies. One way to induce immunological tolerance to allogeneic tissues is via the establishment of mixed hematopoietic chimerism in which the host and donor cells are educated to recognize each other as "self". Proof of principle that in vitro cultured ESC-derived hematopoietic progenitors can be transplanted and induce immunological tolerance to allogeneic tissues exists in mouse models. In this review, we discuss the challenges to in vitro development of a bona fide ESC-derived hematopoietic stem cell and their differentiation fate in vivo, and provide suggestions to predict the immunogenicity of specific ESC-derived hematopoietic populations before transplantation that could be used to prevent their rejection after transplantation into an adult host.

Long-term Culture of Human SSEA-4 Positive Spermatogonial Stem Cells (SSCs)

Recently we and two other groups have shown that human spermatogonial stem cells (SSCs) have the potential to become pluripotent in vitro in defined culture conditions and to differentiate into cells of the three embryonic germ layers. This discovery could open new avenues for autologous cell-based therapy in degenerative diseases, bypassing the ethical and immunological problems related to the human embryonic stem cells. In addition, human SSCs could be used to treat infertility in cancer survival children. However, in order to reprogram SSCs into pluripotency, or to preserve them for repopulation of infertile testes, the first and limiting step is to have access to a highly purified human SSC population that could be multiplied and efficiently cultured in vitro maintaining their molecular and cellular characteristics. Although various studies have attempted to identify molecular markers of human SSCs, to date there is still limited information related to the specific markers that could be used for their isolation and optimized purification that allows long-term in vitro culture of isolated human SSCs. Here using SSEA-4 as an optimal marker for isolation of a subpopulation of SSCs, we show that SSEA-4 positive cells express the highest level of SSC genes compared to other subpopulations isolated with different markers, and can be maintained in culture for over 14 passages which we were unable to obtain with other SSCs markers including GPR125 and ITGA6. In addition, we have established a new technology for cell sorting and long-term culture of human SSC-SSEA-4 positive cells that maximizes the purity and viability of the sorted cells. Our findings are crucial and could be used for the most efficient isolation, purification and long-term culture of SSCs for clinical applications in regenerative medicine, or for preparation of human SSCs for autologous treatment of infertility in cancer survival children.