Cell Therapy For Degenerative Diseases Research Articles

Gene Repair of iPSC Line with GARS (G294R) Mutation of CMT2D Disease by CRISPR/Cas9.

Charcot-Marie-Tooth disease (CMT) severely affects patient activity, and may cause disability. However, no clinical treatment is available to reverse the disease course. The combination of CRISPR/Cas9 and iPSCs may have therapeutic potential against nervous diseases, such as CMT. In the present study, the skin fibroblasts of CMT type 2D (CMT2D) patients with the c.880G>A heterozygous nucleotide mutation in the GARS gene were reprogrammed into iPSCs using three plasmids (pCXLE-hSK, pCXLE-hUL and pCXLE-hOCT3/4-shp5-F). Then, CRISPR/Cas9 technology was used to repair the mutated gene sites at the iPSC level. An iPSC line derived from the GARS (G294R) family with fibular atrophy was successfully induced, and the mutated gene loci were repaired at the iPSC level using CRISPR/Cas9 technology. These findings lay the foundation for future research on drug screening and cell therapy. iPSCs can differentiate into different cell types, and originate from autologous cells. Therefore, they are promising for the development of autologous cell therapies for degenerative diseases. The combination of CRISPR/Cas9 and iPSCs may open a new avenue for the treatment of nervous diseases, such as CMT.

Correlation of micro vessel density and c- Myc expression in breast tumor of mice following mesenchymal stem cell therapy

Stem cell therapy for degenerative diseases has been established; however there are controversies over the treatment of solid tumors with stem cell transplantation. In the present study, the anti-tumor action of mesenchymal stem cells (MSCs) has been examined in a mouse model of breast cancer with emphasize on tumor growth, angiogenesis and c-Myc expression in breast tumors. For this purpose, MSCs were isolated from bone marrow of Balb/c mice and characterized. A Balb/c mouse model of breast cancer was developed and subjected to cell therapy intra venous (I.V) or intra tumor (I.T) with MSCs. Tumor growth was measured using a digital caliber for until the end of experiment (30days). Then the mice were sacrificed and their tumors were removed and processed for histopathological examination, immunohistochemical assay of CD31 and measuring of c-Myc expression using quantitative PCR. Detection of the labeled-MSCs in tumors following injection of the cells (I.V or I.T) clearly showed the homing of MSCs into tumors. Tumor growth in case of MSC-treated mice by I.V and I.T routes was inhibited by approximately 28% and 34% respectively compared to controls. The suppression of angiogenesis was reflected in Micro Vessel Density (MVD) following I.V or I.T delivery of the MSCs. c-Myc gene expression in tumor tissues of mice treated I.V or IT with MSCs was down-regulated to 28.0% and 16.0% respectively compare to control groups. In conclusion, growth inhibition of breast tumors in mice due to MSC therapy is associated with modulation of c-Myc activation and angiogenesis markers.

Clinical potential of human-induced pluripotent stem cells : Perspectives of induced pluripotent stem cells.

The recent establishment of induced pluripotent stem (iPS) cells promises the development of autologous cell therapies for degenerative diseases, without the ethical concerns associated with human embryonic stem (ES) cells. Initially, iPS cells were generated by retroviral transduction of somatic cells with core reprogramming genes. To avoid potential genotoxic effects associated with retroviral transfection, more recently, alternative non-viral gene transfer approaches were developed. Before a potential clinical application of iPS cell-derived therapies can be planned, it must be ensured that the reprogramming to pluripotency is not associated with genome mutagenesis or epigenetic aberrations. This may include direct effects of the reprogramming method or "off-target" effects associated with the reprogramming or the culture conditions. Thus, a rigorous safety testing of iPS or iPS-derived cells is imperative, including long-term studies in model animals. This will include not only rodents but also larger mammalian model species to allow for assessing long-term stability of the transplanted cells, functional integration into the host tissue, and freedom from undifferentiated iPS cells. Determination of the necessary cell dose is also critical; it is assumed that a minimum of 1 billion transplantable cells is required to achieve a therapeutic effect. This will request medium to long-term in vitro cultivation and dozens of cell divisions, bearing the risk of accumulating replication errors. Here, we review the clinical potential of human iPS cells and evaluate which are the most suitable approaches to overcome or minimize risks associated with the application of iPS cell-derived cell therapies.

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.

Non-invasive In-Vivo Imaging of Stem Cells after Transplantation in Cardiovascular Tissue

Stem cell therapy for degenerative diseases, including ischemic heart disease is now a clinical reality. In the search for the optimal cell type for each patient category, many different stem cell subpopulations have been used. In addition, different cell processing procedures and delivery methods have been utilized. Moreover, choices of endpoints have varied between studies. Diverging results have been reported from clinical experiences, with some studies demonstrating promising results with improved cardiac function and reduced mortality and clinical symptoms, and others have seen no improvements. To better understand the underlying mechanisms of these results, a reverse translation from bedside to bench has been opened. Non-invasive cell tracking after implantation has a pivotal role in this translation. Imaging based methods can help elucidate important issues such as retention, migration and efficacy of the transplanted cells. Great effort is being made in finding new and better imaging techniques for different imaging modalities, and much have already been learned. But there are still many unanswered questions. In this review, we give an overview of the imaging modalities used for cell tracking and summarize the latest advances within the field.

Controlling α-SMA expression in adult human pancreatic stem cells by soluble factors

In the application of adult stem cells in regenerative medicine, it is indispensable to control stem cell behaviour in vitro. Since stem cells spontaneously differentiate into several cell types, it is mandatory to identify methods to enrich the desired cell types and concurrently block other differentiation pathways. More precisely, generation of a defined cell population is a key prerequisite for a therapeutic application of stem cells. Here we have demonstrated that it is possible to influence the differentiation of human pancreatic stem cells (hPSCs). During activation of mesodermal differentiation, the cytoskeletal protein alpha-smooth muscle actin (alpha-SMA) seems to play an important role in different cell systems and can usually be detected in hPSCs during in vitro cultivation. We cultured stem cells under different conditions and analyzed the impact on alpha-SMA expression. On the one hand, supplements like retinoic acid (RA) and dimethyl sulfoxide (DMSO) were added to the cultivation medium; on the other hand, different media with or without the addition of fetal calf serum (FCS) were used. Expression of alpha-SMA was determined by immunocytochemistry, Western blot analysis and quantitative RT-PCR. After the treatment of hPSCs with RA, a strong induction of alpha-SMA protein expression was observed when 2mM RA was added to the medium. DMSO in turn induced a marked reduction in alpha-SMA-positive cells. This could also be observed using a keratinocyte serum-free medium (KSFM). Furthermore, the general addition of FCS to the medium had a blocking effect on alpha-SMA expression and decreased the number of alpha-SMA-positive cells to a minimum. The controlled modulation of hPSCs by soluble factors is a first success on the way to a promising application for transplantation medicine and cell therapy of degenerative diseases.

Derivation of engraftable skeletal myoblasts from human embryonic stem cells

Human embryonic stem cells (hESCs) are a promising source for cell therapy in degenerative diseases. A key step in establishing the medical potential of hESCs is the development of techniques for the conversion of hESCs into tissue-restricted precursors suitable for transplantation. We recently described the derivation of multipotent mesenchymal precursors from hESCs. Nevertheless, our previous study was limited by the requirement for mouse feeders and the lack of in vivo data. Here we report a stroma-free induction system for deriving mesenchymal precursors. Selective culture conditions and fluorescence-activated cell sorting (FACS)-mediated purification yielded multipotent mesenchymal precursors and skeletal myoblasts. Skeletal muscle cells undergo in vitro maturation resulting in myotube formation and spontaneous twitching. We found that hESC-derived skeletal myoblasts were viable after transplantation into the tibialis anterior muscle of SCID/Beige mice, as assessed by bioluminescence imaging. Lack of teratoma formation and evidence of long-term myoblast engraftment suggests considerable potential for future therapeutic applications.

Adult pancreatic stem/progenitor cells spontaneously differentiate in vitro into multiple cell lineages and form teratoma-like structures

Cells isolated from pancreas have a remarkable potential for self-renewal and multilineage differentiation. We here present a comprehensive characterisation of stem/progenitor cells derived from exocrine parts of the adult rat pancreas. Using purified cells from either single colonies or even single-cell clones, we specifically demonstrate: (i) the cells contain the typical stem/progenitor cell markers alkaline phophatase, SSEA-1, Oct-4, CD9, Nestin, Pax6, CD44, a-Fetoprotein and Brachyury, demonstrated by immunocytochemistry and RT-PCR; (ii) the cells have the potential to differentiate into lineages of all three germ layers in vitro; (iii) a clonal analysis revealed that even cell lines derived from a single cell have stem/progenitor cell properties such as self-renewal and spontaneous differentiation into various cell lineages; (iv) the cells have the propensity to form three-dimensional, teratoma-like structures in vitro, which contain cells of different lineages; and (v) external stimuli can activate the generation of certain cell types. For instance, cells treated with retinoic acid show an increased expression of alpha-smooth muscle actin. These results suggest that exocrine glands, such as pancreas may be a potential source of adult stem/progenitor cells, suitable for cell therapy of degenerative diseases.

Potential new strategies for the treatment of ovarian infertility and degenerative diseases with autologous ovarian stem cells

The 50-year-old and currently prevailing view that all oocytes in adult human ovaries persist from the fetal period of life is controversial as it clashes with Darwinian evolutionary theory. Studies of oogenesis and follicular renewal in adult human ovaries, and of the role of hormonal signals and third-party cells (tissue macrophages and T cells), could all be helpful in providing better understanding of the causes of ovarian infertility, its prevention and potential therapy. In addition, the authors recently reported differentiation of distinct cell types and the production of new eggs in cultures derived from premenopausal and postmenopausal human ovaries. It is possible that fertilisation of such eggs will open up new opportunities for providing genetically related children to infertile women for whom conventional in vitro fertilisation has failed. As ovarian stem cells appear to represent a new type of totipotent adult stem cell, they could also be utilised for autologous stem cell therapy of degenerative diseases, without any involvement of allogeneic embryonic stem cells and somatic cell nuclear transfer.

Human embryonic stem cells: challenges and opportunities

Human and non-human primate embryonic stem (ES) cells are invaluable resources for developmental studies, pharmaceutical research and a better understanding of human disease and replacement therapies. In 1998, subsequent to the establishment of the first monkey ES cell line in 1995, the first human ES cell line was developed. Later, three of the National Institute of Health (NIH) lines (BG01, BG02 and BG03) were derived from embryos that would have been discarded because of their poor quality. A major challenge to research in this area is maintaining the unique characteristics and a normal karyotype in the NIH-registered human ES cell lines. A normal karyotype can be maintained under certain culture conditions. In addition, a major goal in stem cell research is to direct ES cells towards a limited cell fate, with research progressing towards the derivation of a variety of cell types. We and others have built on findings in vertebrate (frog, chicken and mouse) neural development and from mouse ES cell research to derive neural stem cells from human ES cells. We have directed these derived human neural stem cells to differentiate into motoneurons using a combination of developmental cues (growth factors) that are spatially and temporally defined. These and other human ES cell derivatives will be used to screen new compounds and develop innovative cell therapies for degenerative diseases.