Lithium chloride improves the efficiency of induced pluripotent stem cell-derived neurospheres.

Induced pluripotent stem cell (iPSC)-derived neurospheres, which consist mainly of neural progenitors, are considered to be a good source of neural cells for transplantation in regenerative medicine. In this study, we have used lithium chloride, which is known to be a neuroprotective agent, in an iPSC-derived neurosphere model, and examined both the formation rate and size of the neurospheres as well as the proliferative and apoptotic status of their contents. Our results showed that lithium enhanced the formation and the sizes of the iPSC-derived neurospheres, increased the number of Ki67-positive proliferating cells, but reduced the number of the TUNEL-positive apoptotic cells. This increased number of Ki67 proliferating cells was secondary to the decreased apoptosis and not to the stimulation of cell cycle entry, as the expression of the proliferation marker cyclin D1 mRNA did not change after lithium treatment. Altogether, we suggest that lithium enhances the survival of neural progenitors and thus the quality of the iPSC-derived neurospheres, which may strengthen the prospect of using lithium-treated pluripotent cells and their derivatives in a clinical setting.

Application of human induced pluripotent stem cells for modeling and treating neurodegenerative diseases.

The advent of human induced pluripotent stem cells (hiPSCs), reprogrammed in vitro from both healthy and disease-state human somatic cells, has triggered an enormous global research effort to realize personalized regenerative medicine for numerous degenerative conditions. hiPSCs have been generated from cells of many tissue types and can be differentiated in vitro to most somatic lineages, not only for the establishment of disease models that can be utilized as novel drug screening platforms and to study the molecular and cellular processes leading to degeneration, but also for the in vivo cell-based repair or modulation of a patient's disease profile. hiPSCs derived from patients with the neurodegenerative diseases amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease and multiple sclerosis have been successfully differentiated in vitro into disease-relevant cell types, including motor neurons, dopaminergic neurons and oligodendrocytes. However, the generation of functional iPSC-derived neural cells that are capable of engraftment in humans and the identification of robust disease phenotypes for modeling neurodegeneration still require several key challenges to be addressed. Here, we discuss these challenges and summarize recent progress toward the application of iPSC technology for these four common neurodegenerative diseases.

Fine structure of neurally differentiated iPS cells generated from a multiple sclerosis (MS) patient: a case study.

We compared the characteristics of neural cells derived from induced pluripotent stem (iPS) cells from a patient with multiple sclerosis versus neurally differentiated control iPS cells of a healthy individual. The iPS cells were differentiated toward the oligodendrocyte lineage using a four-step protocol established for the differentiation of embryonic stem cells. The resulting cell population was immunostained on day 112 of differentiation for the presence of oligodendrocytes and analyzed by transmission electron microscopy (TEM). Both patient and control samples resembled a mixed population of neural cells rather than oligodendroglia of high purity, including neural stem cell-like cells and possibly oligodendrocytes demonstrable by TEM.

Distinct immunomodulatory and migratory mechanisms underpin the therapeutic potential of human mesenchymal stem cells in autoimmune demyelination.

Mesenchymal stem cells (MSCs) are efficacious in a variety of intractable diseases. While bone marrow (BM)-derived MSCs (BM-MSCs) have been widely investigated, MSCs from other tissue sources have also been shown to be effective in several autoimmune and inflammatory disorders. In the present study, we simultaneously assessed the therapeutic efficacy of human BM-MSCs, as well as MSCs isolated from adipose tissue (Ad-MSCs) and umbilical cord Wharton's jelly (UC-MSCs), in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Prior to in vivo experiments, we characterized the phenotype and function of all three MSC types. We show that BM-MSCs were more efficient at suppressing the in vitro proliferation of mitogen or antigen-stimulated T-cell responses compared to Ad-MSCs and UC-MSCs. Notably BM-MSCs induced the differential expression of cytokines from normal and stimulated T-cells. Paradoxically, intravenous transplantation of BM-MSCs into C57Bl/6 mice with chronic progressive EAE had a negligible effect on the disease course, even when multiple MSC injections were administered over a number of time points. In contrast, Ad-MSCs had the most significant impact on clinical and pathological disease outcomes in chronic progressive and relapsing-remitting EAE models. In vivo tracking studies revealed that Ad-MSCs were able to migrate to the central nervous system (CNS), a property that most likely correlated with their broader expression of homing molecules, while BM-MSCs were not detected in this anatomic region. Collectively, this comparative investigation demonstrates that transplanted Ad-MSCs play a significant role in tissue repair processes by virtue of their ability to suppress inflammation coupled with their enhanced ability to home to the injured CNS. Given the access and relatively ease for harvesting adipose tissue, these data further implicate Ad-MSCs as a cell therapeutic that may be used to treat MS patients.

Early intervention with gene-modified mesenchymal stem cells overexpressing interleukin-4 enhances anti-inflammatory responses and functional recovery in experimental autoimmune demyelination.

Mesenchymal stem/stromal cells (MSCs) can be isolated from most adult tissues and hold considerable promise for tissue regenerative therapies. Some of the potential advantages that MSCs have over other adult stem cell types include: (1) their relative ease of isolation, culture and expansion; (2) their immunomodulatory properties; (3) they can provide trophic support to injured tissues; (4) they can be transduced by retroviral vectors at a high efficiency; (5) they have an ability to home to sites of inflammation and injury. Collectively these characteristics suggest that MSCs are attractive vehicles for cell and gene therapy applications. In the current study, we investigated whether transplantation of human adipose-derived MSCs (Ad-MSCs) engineered to overexpress the anti-inflammatory cytokine interleukin (IL)-4 was efficacious in experimental autoimmune encephalomyelitis (EAE). Ad-MSCs transduced with a bicistronic lentiviral vector encoding mouse IL-4 and enhanced green fluorescent protein (Ad-IL4-MSCs) stably expressed, relatively high levels of both transgenes. Importantly the phenotypic and functional attributes of Ad-IL4-MSCs, such as the expression of homing molecules and differentiation capacity, was not altered by the transduction process. Notably, the early administration of Ad-IL4-MSCs in mice with EAE at the time of T-cell priming attenuated clinical disease. This protective effect was associated with a reduction in peripheral MOG-specific T-cell responses and a shift from a pro- to an anti-inflammatory cytokine response. These data suggest that the delivery of Ad-MSCs genetically engineered to express anti-inflammatory cytokines may provide a rational approach to promote immunomodulation and tissue protection in a number of inflammatory and degenerative diseases including multiple sclerosis.

Comparative study on the therapeutic potential of neurally differentiated stem cells in a mouse model of multiple sclerosis.

BACKGROUND: Transplantation of neural stem cells (NSCs) is a promising novel approach to the treatment of neuroinflammatory diseases such as multiple sclerosis (MS). NSCs can be derived from primary central nervous system (CNS) tissue or obtained by neural differentiation of embryonic stem (ES) cells, the latter having the advantage of readily providing an unlimited number of cells for therapeutic purposes. Using a mouse model of MS, we evaluated the therapeutic potential of NSCs derived from ES cells by two different neural differentiation protocols that utilized adherent culture conditions and compared their effect to primary NSCs derived from the subventricular zone (SVZ). METHODOLOGY/PRINCIPAL FINDINGS: The proliferation and secretion of pro-inflammatory cytokines by antigen-stimulated splenocytes was reduced in the presence of SVZ-NSCs, while ES cell-derived NSCs exerted differential immunosuppressive effects. Surprisingly, intravenously injected NSCs displayed no significant therapeutic impact on clinical and pathological disease outcomes in mice with experimental autoimmune encephalomyelitis (EAE) induced by recombinant myelin oligodendrocyte glycoprotein, independent of the cell source. Studies tracking the biodistribution of transplanted ES cell-derived NSCs revealed that these cells were unable to traffic to the CNS or peripheral lymphoid tissues, consistent with the lack of cell surface homing molecules. Attenuation of peripheral immune responses could only be achieved through multiple high doses of NSCs administered intraperitoneally, which led to some neuroprotective effects within the CNS. CONCLUSION/SIGNIFICANCE: Systemic transplantation of these NSCs does not have a major influence on the clinical course of rMOG-induced EAE. Improving the efficiency at which NSCs home to inflammatory sites may enhance their therapeutic potential in this model of CNS autoimmunity.

Neural differentiation of patient specific iPS cells as a novel approach to study the pathophysiology of multiple sclerosis.

The recent introduction of technologies capable of reprogramming human somatic cells into induced pluripotent stem (iPS) cells offers a unique opportunity to study many aspects of neurodegenerative diseases in vitro that could ultimately lead to novel drug development and testing. Here, we report for the first time that human dermal fibroblasts from a patient with relapsing-remitting Multiple Sclerosis (MS) were reprogrammed to pluripotency by retroviral transduction using defined factors (OCT4, SOX2, KLF4, and c-MYC). The MSiPS cell lines resembled human embryonic stem (hES) cell-like colonies in morphology and gene expression and exhibited silencing of the retroviral transgenes after four passages. MSiPS cells formed embryoid bodies that expressed markers of all three germ layers by immunostaining and Reverse Transcriptase (RT)-PCR. The injection of undifferentiated iPS cell colonies into immunodeficient mice formed teratomas, thereby demonstrating pluripotency. The MSiPS cells were successfully differentiated into mature astrocytes, oligodendrocytes and neurons with normal karyotypes. Although MSiPS-derived neurons displayed some differences in their electrophysiological characteristics as compared to the control cell line, they exhibit properties of functional neurons, with robust resting membrane potentials, large fast tetrodotoxin-sensitive action potentials and voltage-gated sodium currents. This study provides for the first time proof of concept that disease cell lines derived from skin cells obtained from an MS patient can be generated and successfully differentiated into mature neural lineages. This represents an important step in a novel approach for the study of MS pathophysiology and potential drug discovery.

CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells.

The application of human embryonic stem (hES) cells in regenerative medicine will require rigorous quality control measures to ensure the safety of hES cell-derived grafts. During propagation in vitro, hES cells can acquire cytogenetic abnormalities as well as submicroscopic genetic lesions, such as small amplifications or deletions. Many of the genetic abnormalities that arise in hES cell cultures are also implicated in human cancer development. The causes of genetic instability of hES cells in culture are poorly understood, and commonly used cytogenetic methods for detection of abnormal cells are capable only of low-throughput analysis on small numbers of cells. The identification of biomarkers of genetic instability in hES cells would greatly facilitate the development of culture methods that preserve genomic integrity. Here we show that CD30, a member of the tumor necrosis factor receptor superfamily, is expressed on transformed but not normal hES cells, and that CD30 expression protects hES cells against apoptosis.