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.

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.

Human breast tumor cells induce self-tolerance mechanisms to avoid NKG2D-mediated and DNAM-mediated NK cell recognition.

Breast cancer is the leading cause of death for women between the ages of 35 to 65. This is mostly due to intertumor heterogeneity and the lack of specific therapies for all subtypes. However, some breast cancers with an unexpected good prognosis are associated with enhanced antitumor immunity in situ. We studied whether breast cancer subtypes might have different susceptibilities to natural killer (NK) cells' antitumor immunity. We collected a large public set of microarray data for primary breast tumors and determined NK cell ligand expression. We found that despite heterogeneous levels of inhibitory HLA members, NKG2D ligands and DNAM ligands are expressed in virtually all breast tumor subtypes. Functional experiments in breast cancer subtypes expressing various levels of NK cell ligands showed that NK-mediated cytotoxicity is mainly HLA, NKG2D, and DNAM dependent. In parallel, we showed that cell lines and primary breast tumor cells secrete soluble inhibitory factors that alter NK cell functions. Finally, we showed that these mechanisms of escape occur in vivo in the MMTV-Neu model of spontaneous murine breast cancer. Our study shows that breast cancer cells, independent of the subtype, have developed different mechanisms to escape from NK cells' antitumor immunity. These results emphasize the role of NK cells in breast tumor clearance and underlie the importance of devising future therapy aiming at enhancing NK cell-mediated recognition in parallel with the prevention of the tumor-editing process.

Differential role for CD277 as a co-regulator of the immune signal in T and NK cells.

The human butyrophilin (BTN) 3 or CD277 molecules belong to the B7 family members and are expressed in various immune cells such as T and NK cells. Here, we show that CD277 triggering considerably enhances TCR-induced cytokine production and cell proliferation, even when another co-stimulatory molecule, CD28, is engaged. These CD277-induced additive functional effects are in accordance with the detection of early T-cell activation events such as TCR-induced cell signaling being increased upon CD277 engagement. However, we found that CD277 triggering is not involved in CD16- or NKp46-induced NK cell activation. BTN3/CD277 comprises three structurally related members, BTN3A1, BTN3A2 and BTN3A3. CD277 antibodies recognize all isoforms and we describe a differential expression of BTN3 isoforms between T and NK cells that could explain differential CD277 functions between T and NK cells. Our results show that, while T cells express all BTN3/CD277 transcripts, NK cells express mostly BTN3A2, which lacks the B30.2 intracellular domain. Furthermore, NKp30-induced cytokine production is decreased by the specific engagement of BTN3A2, but not by BTN3A1 triggering. Thus, we provide new insights into the CD277 co-stimulatory pathway that may differentially participate in the regulation of various cell-mediated immune responses.

Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity.

NK cells are a major component of the antitumor immune response and are involved in controlling tumor progression and metastases in animal models. Here, we show that dysfunction of these cells accompanies human breast tumor progression. We characterized human peripheral blood NK (p-NK) cells and malignant mammary tumor-infiltrating NK (Ti-NK) cells from patients with noninvasive and invasive breast cancers. NK cells isolated from the peripheral blood of healthy donors and normal breast tissue were used as controls. With disease progression, we found that expression of activating NK cell receptors (such as NKp30, NKG2D, DNAM-1, and CD16) decreased while expression of inhibitory receptors (such as NKG2A) increased and that this correlated with decreased NK cell function, most notably cytotoxicity. Importantly, Ti-NK cells had more pronounced impairment of their cytotoxic potential than p-NK cells. We also identified several stroma-derived factors, including TGF-ß1, involved in tumor-induced reduction of normal NK cell function. Our data therefore show that breast tumor progression involves NK cell dysfunction and that breast tumors model their environment to evade NK cell antitumor immunity. This highlights the importance of developing future therapies able to restore NK cell cytotoxicity to limit/prevent tumor escape from antitumor immunity.

Generation of induced pluripotent stem cells from human kidney mesangial cells.

Glomerular injury and podocyte loss leads to secondary tubulointerstitial damage and the development of fibrosis. The possibility of genetically reprogramming adult cells, termed induced pluripotent stem cells (iPS), may pave the way for patient-specific stem-cell-based therapies. Here, we reprogrammed normal human mesangial cells to pluripotency by retroviral transduction using defined factors (OCT4, SOX2, KLF4 and c-Myc). The kidney iPS (kiPS) cells resembled human embryonic stem-cell-like colonies in morphology and gene expression: They were alkaline phosphatase-positive; expressed OCT3/4, TRA-1 to 60 and TRA-1 to 81 proteins; and showed downregulation of mesangial cell markers. Quantitative (qPCR) showed that kiPS cells expressed genes analogous to embryonic stem cells and exhibited silencing of the retroviral transgenes by the fourth passage of differentiation. Furthermore, kiPS cells formed embryoid bodies and expressed markers of all three germ layers. The injection of undifferentiated kiPS colonies into immunodeficient mice formed teratomas, thereby demonstrating pluripotency. These results suggest that reprogrammed kidney induced pluripotent stem cells may aid the study of genetic kidney diseases and lead to the development of novel therapies.