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.

Challenges of translating a cell therapy to GMP.

Over the last decade, cell and gene therapies have contributed remarkably to the array of novel therapies combating diseases that did not have any hope for an effective treatment or, let alone, a cure. This remarkable achievement was underlined by the marketing approval of CAR T cell therapies in 2017 in the United States, followed by many other countries, world-wide. Since then, thousands of patients have benefited from this autologous, gene modified cell therapy (Abou-El-Enein et al., 2021). Rare diseases, particularly innate neurological diseases such as Huntington's disease have also been a target for cell therapies. The notion of being able to augment or replace the function of diseased neurons with progenitor cells or neurons derived from human stem cells has been researched for the last 10 years and is finally reaching the stage of clinical translation (Holley et al., 2018; Reidling et al., 2018). With these cellular and gene therapies reaching clinical applicability, it is important to bring them to patients in a safe, efficacious and reliable way, and for this purpose, Good Manufacturing Practice (GMP) needs to be applied to the manufacturing of such novel and often life-saving therapies. In the first decade of the 21st century, gene therapies, particularly in vivo adenoviral vector gene therapy (Wilson, 2009) and hematopoietic stem cell gene therapies (Hacein-Bey-Abina et al., 2008) were associated with adverse events that were highly publicized and gave the field a bad reputation in the public eye. The last two decades, however, due to the meticulous work of dedicated researchers, and excellent progress in GMP manufacturing, cell and gene therapies have become safe and efficacious and have propelled the field to the forefront of the most promising novel therapies available for current unmet medical needs. This book chapter will discuss the historical perspective of cellular therapies and their development, will describe the currently available cell and gene therapies for different diseases and their GMP manufacturing methods and challenges, and will point out the future direction of these therapies and their envisioned manufacturing, as can be foreseen currently.

Target receptor identification and subsequent treatment of resected brain tumors with encapsulated and engineered allogeneic stem cells.

Cellular therapies offer a promising therapeutic strategy for the highly malignant brain tumor, glioblastoma (GBM). However, their clinical translation is limited by the lack of effective target identification and stringent testing in pre-clinical models that replicate standard treatment in GBM patients. In this study, we show the detection of cell surface death receptor (DR) target on CD146-enriched circulating tumor cells (CTC) captured from the blood of mice bearing GBM and patients diagnosed with GBM. Next, we developed allogeneic "off-the-shelf" clinical-grade bifunctional mesenchymal stem cells (MSCBif) expressing DR-targeted ligand and a safety kill switch. We show that biodegradable hydrogel encapsulated MSCBif (EnMSCBif) has a profound therapeutic efficacy in mice bearing patient-derived invasive, primary and recurrent GBM tumors following surgical resection. Activation of the kill switch enhances the efficacy of MSCBif and results in their elimination post-tumor treatment which can be tracked by positron emission tomography (PET) imaging. This study establishes a foundation towards a clinical trial of EnMSCBif in primary and recurrent GBM patients.

Primed mesenchymal stem cells package exosomes with metabolites associated with immunomodulation.

Mesenchymal stem cell (MSC) based therapies are currently being evaluated as a putative therapeutic in numerous human clinical trials. Recent reports have established that exosomes mediate much of the therapeutic properties of MSCs. Exosomes are nanovesicles which mediate intercellular communication, transmitting signals between cells which regulate a diverse range of biological processes. MSC-derived exosomes are packaged with numerous types of proteins and RNAs, however, their metabolomic and lipidomic profiles to date have not been well characterized. We previously reported that MSCs, in response to priming culture conditions that mimic the in vivo microenvironmental niche, substantially modulate cellular signaling and significantly increase the secretion of exosomes. Here we report that MSCs exposed to such priming conditions undergo glycolytic reprogramming, which homogenizes MSCs' metabolomic profile. In addition, we establish that exosomes derive from primed MSCs are packaged with numerous metabolites that have been directly associated with immunomodulation, including M2 macrophage polarization and regulatory T lymphocyte induction.

Exosomes Derived from Human Primed Mesenchymal Stem Cells Induce Mitosis and Potentiate Growth Factor Secretion.

Mesenchymal stem cells (MSCs) facilitate functional recovery in numerous animal models of inflammatory and ischemic tissue-related diseases with a growing body of research suggesting that exosomes mediate many of these therapeutic effects. It remains unclear, however, which types of proteins are packaged into exosomes compared with the cells from which they are derived. In this study, using comprehensive proteomic analysis, we demonstrated that human primed MSCs secrete exosomes (pMEX) that are packaged with markedly higher fractions of specific protein subclasses compared with their cells of origin, indicating regulation of their contents. Notably, we found that pMEX are also packaged with substantially elevated levels of extracellular-associated proteins. Fibronectin was the most abundant protein detected, and data established that fibronectin mediates the mitogenic properties of pMEX. In addition, treatment of SHSY5Y cells with pMEX induced the secretion of growth factors known to possess mitogenic and neurotrophic properties. Taken together, our comprehensive analysis indicates that pMEX are packaged with specific protein subtypes, which may provide a molecular basis for their distinct functional properties.

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.

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.

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.

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.

Comprehensive Proteomic Analysis of Mesenchymal Stem Cell Exosomes Reveals Modulation of Angiogenesis via Nuclear Factor-KappaB Signaling.

Mesenchymal stem cells (MSC) are known to facilitate healing of ischemic tissue related diseases through proangiogenic secretory proteins. Recent studies further show that MSC derived exosomes function as paracrine effectors of angiogenesis, however, the identity of which components of the exosome proteome responsible for this effect remains elusive. To address this we used high-resolution isoelectric focusing coupled liquid chromatography tandem mass spectrometry, an unbiased high throughput proteomics approach to comprehensively characterize the proteinaceous contents of MSCs and MSC derived exosomes. We probed the proteome of MSCs and MSC derived exosomes from cells cultured under expansion conditions and under ischemic tissue simulated conditions to elucidate key angiogenic paracrine effectors present and potentially differentially expressed in these conditions. In total, 6,342 proteins were identified in MSCs and 1,927 proteins in MSC derived exosomes, representing to our knowledge the first time these proteomes have been probed comprehensively. Multilayered analyses identified several putative paracrine effectors of angiogenesis present in MSC exosomes and increased in expression in MSCs exposed to ischemic tissue-simulated conditions; these include platelet derived growth factor, epidermal growth factor, fibroblast growth factor, and most notably nuclear factor-kappaB (NFkB) signaling pathway proteins. NFkB signaling was identified as a key mediator of MSC exosome induced angiogenesis in endothelial cells by functional in vitro validation using a specific inhibitor. Collectively, the results of our proteomic analysis show that MSC derived exosomes contain a robust profile of angiogenic paracrine effectors, which have potential for the treatment of ischemic tissue-related diseases.

Human Mesenchymal Stem Cells Genetically Engineered to Overexpress Brain-derived Neurotrophic Factor Improve Outcomes in Huntington's Disease Mouse Models.

Huntington's disease (HD) is a fatal degenerative autosomal dominant neuropsychiatric disease that causes neuronal death and is characterized by progressive striatal and then widespread brain atrophy. Brain-derived neurotrophic factor (BDNF) is a lead candidate for the treatment of HD, as it has been shown to prevent cell death and to stimulate the growth and migration of new neurons in the brain in transgenic mouse models. BDNF levels are reduced in HD postmortem human brain. Previous studies have shown efficacy of mesenchymal stem/stromal cells (MSC)/BDNF using murine MSCs, and the present study used human MSCs to advance the therapeutic potential of the MSC/BDNF platform for clinical application. Double-blinded studies were performed to examine the effects of intrastriatally transplanted human MSC/BDNF on disease progression in two strains of immune-suppressed HD transgenic mice: YAC128 and R6/2. MSC/BDNF treatment decreased striatal atrophy in YAC128 mice. MSC/BDNF treatment also significantly reduced anxiety as measured in the open-field assay. Both MSC and MSC/BDNF treatments induced a significant increase in neurogenesis-like activity in R6/2 mice. MSC/BDNF treatment also increased the mean lifespan of the R6/2 mice. Our genetically modified MSC/BDNF cells set a precedent for stem cell-based neurotherapeutics and could potentially be modified for other neurodegenerative disorders such as amyotrophic lateral sclerosis, Alzheimer's disease, and some forms of Parkinson's disease. These cells provide a platform delivery system for future studies involving corrective gene-editing strategies.

Large-scale production of lentiviral vector in a closed system hollow fiber bioreactor.

Lentiviral vectors are widely used in the field of gene therapy as an effective method for permanent gene delivery. While current methods of producing small scale vector batches for research purposes depend largely on culture flasks, the emergence and popularity of lentiviral vectors in translational, preclinical and clinical research has demanded their production on a much larger scale, a task that can be difficult to manage with the numbers of producer cell culture flasks required for large volumes of vector. To generate a large scale, partially closed system method for the manufacturing of clinical grade lentiviral vector suitable for the generation of induced pluripotent stem cells (iPSCs), we developed a method employing a hollow fiber bioreactor traditionally used for cell expansion. We have demonstrated the growth, transfection, and vector-producing capability of 293T producer cells in this system. Vector particle RNA titers after subsequent vector concentration yielded values comparable to lentiviral iPSC induction vector batches produced using traditional culture methods in 225 cm(2) flasks (T225s) and in 10-layer cell factories (CF10s), while yielding a volume nearly 145 times larger than the yield from a T225 flask and nearly three times larger than the yield from a CF10. Employing a closed system hollow fiber bioreactor for vector production offers the possibility of manufacturing large quantities of gene therapy vector while minimizing reagent usage, equipment footprint, and open system manipulation.

Generation of HIV-1 resistant and functional macrophages from hematopoietic stem cell-derived induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. By developing iPSCs to treat HIV, there is the potential for generating a continuous supply of therapeutic cells for transplantation into HIV-infected patients. In this study, we have used human hematopoietic stem cells (HSCs) to generate anti-HIV gene expressing iPSCs for HIV gene therapy. HSCs were dedifferentiated into continuously growing iPSC lines with four reprogramming factors and a combination anti-HIV lentiviral vector containing a CCR5 short hairpin RNA (shRNA) and a human/rhesus chimeric TRIM5α gene. Upon directed differentiation of the anti-HIV iPSCs toward the hematopoietic lineage, a robust quantity of colony-forming CD133(+) HSCs were obtained. These cells were further differentiated into functional end-stage macrophages which displayed a normal phenotypic profile. Upon viral challenge, the anti-HIV iPSC-derived macrophages exhibited strong protection from HIV-1 infection. Here, we demonstrate the ability of iPSCs to develop into HIV-1 resistant immune cells and highlight the potential use of iPSCs for HIV gene and cellular therapies.