Transplanted human neural stem cells rescue phenotypes in zQ175 Huntington's disease mice and innervate the striatum.

Huntington's disease (HD), a genetic neurodegenerative disorder, primarily affects the striatum and cortex with progressive loss of medium-sized spiny neurons (MSNs) and pyramidal neurons, disrupting cortico-striatal circuitry. A promising regenerative therapeutic strategy of transplanting human neural stem cells (hNSCs) is challenged by the need for long-term functional integration. We previously described that, with short-term hNSC transplantation into the striatum of HD R6/2 mice, human cells differentiated into electrophysiologically active immature neurons, improving behavior and biochemical deficits. Here, we show that long-term (8 months) implantation of hNSCs into the striatum of HD zQ175 mice ameliorates behavioral deficits, increases brain-derived neurotrophic factor (BDNF) levels, and reduces mutant huntingtin (mHTT) accumulation. Patch clamp recordings, immunohistochemistry, single-nucleus RNA sequencing (RNA-seq), and electron microscopy demonstrate that hNSCs differentiate into diverse neuronal populations, including MSN- and interneuron-like cells, and form connections. Single-nucleus RNA-seq analysis also shows restoration of several mHTT-mediated transcriptional changes of endogenous striatal HD mouse cells. Remarkably, engrafted cells receive synaptic inputs, innervate host neurons, and improve membrane and synaptic properties. Overall, the findings support hNSC transplantation for further evaluation and clinical development for HD.

Mesenchymal stem/stromal cells genetically engineered to produce vascular endothelial growth factor for revascularization in wound healing and ischemic conditions.

Mesenchymal stem/stromal cells (MSCs) may be able to improve ischemic conditions as they can actively seek out areas of low oxygen and secrete proangiogenic factors. In more severe trauma and chronic cases, however, cells alone may not be enough. Therefore, we have combined the stem cell and angiogenic factor approaches to make a more potent therapy. We developed an engineered stem cell therapy product designed to treat critical limb ischemia that could also be used in trauma-induced scarring and fibrosis where additional collateral blood flow is needed following damage to and blockage of the primary vessels. We used MSCs from normal human donor marrow and engineered them to produce high levels of the angiogenic factor vascular endothelial growth factor (VEGF). The MSC/VEGF product has been successfully developed and characterized using good manufacturing practice (GMP)-compliant methods, and we have completed experiments showing that MSC/VEGF significantly increased blood flow in the ischemic limb of immune deficient mice, compared to the saline controls in each study. We also performed safety studies demonstrating that the injected product does not cause harm and that the cells remain around the injection site for more than 1 month after hypoxic preconditioning. An on-demand formulation system for delivery of the product to clinical sites that lack cell processing facilities is in development.

Highly Efficient Differentiation of Endothelial Cells from Pluripotent Stem Cells Requires the MAPK and the PI3K Pathways.

Pluripotent stem cells are a promising source of endothelial cells (ECs) for the treatment of vascular diseases. We have developed a robust protocol to differentiate human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) into ECs with high purities (94%-97% CD31+ and 78%-83% VE-cadherin+ ) in 8 days without cell sorting. Passaging of these cells yielded a nearly pure population of ECs (99% of CD31+ and 96.8% VE-cadherin+ ). These ECs also expressed other endothelial markers vWF, Tie2, NOS3, and exhibited functions of ECs such as uptake of Dil-acetylated low-density lipoprotein and formation of tubes in vitro or vessels in vivo on matrigel. We found that FGF2, VEGF, and BMP4 synergistically induced early vascular progenitors (VPs) from hiPSC-derived mesodermal cells. The MAPK and PI3K pathways are crucial not only for the initial commitment to vascular lineages but also for the differentiation of vascular progenitors to ECs, most likely through regulation of the ETS family transcription factors, ERG and FLI1. We revealed novel roles of the p38 and JNK MAPK pathways on EC differentiation. Furthermore, inhibition of the ERK pathway markedly promoted the differentiation of smooth muscle cells. Finally, we demonstrate that pluripotent stem cell-derived ECs are capable of forming patent blood vessels that were connected to the host vasculature in the ischemic limbs of immune deficient mice. Thus, we demonstrate that ECs can be efficiently derived from hiPSCs and hESCs, and have great potential for vascular therapy as well as for mechanistic studies of EC differentiation. Stem Cells 2017;35:909-919.

Therapeutic effects of stem cells in rodent models of Huntington's disease: Review and electrophysiological findings.

The principal symptoms of Huntington's disease (HD), chorea, cognitive deficits, and psychiatric symptoms are associated with the massive loss of striatal and cortical projection neurons. As current drug therapies only partially alleviate symptoms, finding alternative treatments has become peremptory. Cell replacement using stem cells is a rapidly expanding field that offers such an alternative. In this review, we examine recent studies that use mesenchymal cells, as well as pluripotent, cell-derived products in animal models of HD. Additionally, we provide further electrophysiological characterization of a human neural stem cell line, ESI-017, which has already demonstrated disease-modifying properties in two mouse models of HD. Overall, the field of regenerative medicine represents a viable and promising avenue for the treatment of neurodegenerative disorders including HD.

SV-BR-1-GM, a Clinically Effective GM-CSF-Secreting Breast Cancer Cell Line, Expresses an Immune Signature and Directly Activates CD4+ T Lymphocytes.

Targeted cancer immunotherapy with irradiated, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting, allogeneic cancer cell lines has been an effective approach to reduce tumor burden in several patients. It is generally assumed that to be effective, these cell lines need to express immunogenic antigens coexpressed in patient tumor cells, and antigen-presenting cells need to take up such antigens then present them to patient T cells. We have previously reported that, in a phase I pilot study (ClinicalTrials.gov NCT00095862), a subject with stage IV breast cancer experienced substantial regression of breast, lung, and brain lesions following inoculation with clinical formulations of SV-BR-1-GM, a GM-CSF-secreting breast tumor cell line. To identify diagnostic features permitting the prospective identification of patients likely to benefit from SV-BR-1-GM, we conducted a molecular analysis of the SV-BR-1-GM cell line and of patient-derived blood, as well as a tumor specimen. Compared to normal human breast cells, SV-BR-1-GM cells overexpress genes encoding tumor-associated antigens (TAAs) such as PRAME, a cancer/testis antigen. Curiously, despite its presumptive breast epithelial origin, the cell line expresses major histocompatibility complex (MHC) class II genes (HLA-DRA, HLA-DRB3, HLA-DMA, HLA-DMB), in addition to several other factors known to play immunostimulatory roles. These factors include MHC class I components (B2M, HLA-A, HLA-B), ADA (encoding adenosine deaminase), ADGRE5 (CD97), CD58 (LFA3), CD74 (encoding invariant chain and CLIP), CD83, CXCL8 (IL8), CXCL16, HLA-F, IL6, IL18, and KITLG. Moreover, both SV-BR-1-GM cells and the responding study subject carried an HLA-DRB3*02:02 allele, raising the question of whether SV-BR-1-GM cells can directly present endogenous antigens to T cells, thereby inducing a tumor-directed immune response. In support of this, SV-BR-1-GM cells (which also carry the HLA-DRB3*01:01 allele) treated with yellow fever virus (YFV) envelope (Env) 43-59 peptides reactivated YFV-DRB3*01:01-specific CD4+ T cells. Thus, the partial HLA allele match between SV-BR-1-GM and the clinical responder might have enabled patient T lymphocytes to directly recognize SV-BR-1-GM TAAs as presented on SV-BR-1-GM MHCs. Taken together, our findings are consistent with a potentially unique mechanism of action by which SV-BR-1-GM cells can act as APCs for previously primed CD4+ T cells.

Human Neural Stem Cell Transplantation Rescues Functional Deficits in R6/2 and Q140 Huntington's Disease Mice.

Huntington's disease (HD) is an inherited neurodegenerative disorder with no disease-modifying treatment. Expansion of the glutamine-encoding repeat in the Huntingtin (HTT) gene causes broad effects that are a challenge for single treatment strategies. Strategies based on human stem cells offer a promising option. We evaluated efficacy of transplanting a good manufacturing practice (GMP)-grade human embryonic stem cell-derived neural stem cell (hNSC) line into striatum of HD modeled mice. In HD fragment model R6/2 mice, transplants improve motor deficits, rescue synaptic alterations, and are contacted by nerve terminals from mouse cells. Furthermore, implanted hNSCs are electrophysiologically active. hNSCs also improved motor and late-stage cognitive impairment in a second HD model, Q140 knockin mice. Disease-modifying activity is suggested by the reduction of aberrant accumulation of mutant HTT protein and expression of brain-derived neurotrophic factor (BDNF) in both models. These findings hold promise for future development of stem cell-based therapies.