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Dopaminergic neurons are the predominant brain cells affected in Parkinson's disease. With the limited availability of live human brain dopaminergic neurons to study pathological mechanisms of Parkinson's disease, dopaminergic neurons have been generated from human-skin-cell-derived induced pluripotent stem cells. Originally, induced pluripotent stem-cell-derived dopaminergic neurons were generated using small molecules. These neurons took more than two months to mature. However, the transcription-factor-mediated differentiation of induced pluripotent stem cells has revealed quicker and cheaper methods to generate dopaminergic neurons. In this study, we compared and contrasted three protocols to generate induced pluripotent stem-cell-derived dopaminergic neurons using transcription-factor-mediated directed differentiation. We deviated from the established protocols using lentivirus transduction to stably integrate different transcription factors into the AAVS1 safe harbour locus of induced pluripotent stem cells. We used different media compositions to generate more than 90% of neurons in the culture, out of which more than 85% of the neurons were dopaminergic neurons within three weeks. Therefore, from our comparative study, we reveal that a combination of transcription factors along with small molecule treatment may be required to generate a pure population of human dopaminergic neurons.

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Genetically encoded calcium indicators (GECIs) and high-resolution confocal microscopy enable dynamic visualization of calcium signals in cells and tissues. Two-dimensional and 3D biocompatible materials mimic the mechanical microenvironments of tumor and healthy tissues in a programmable manner. Cancer xenograft models and ex vivo functional imaging of tumor slices reveal physiologically relevant functions of calcium dynamics in tumors at different progression stages. Integration of these powerful techniques allows us to quantify, diagnose, model, and understand cancer pathobiology. Here, we describe detailed materials and methods used to establish this integrated interrogation platform, from generating transduced cancer cell lines that stably express CaViar (GCaMP5G + QuasAr2) to in vitro and ex vivo calcium imaging of the cells in 2D/3D hydrogels and tumor tissues. These tools open the possibility for detailed explorations of mechano-electro-chemical network dynamics in living systems.

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Heparin, a highly sulfated and epimerized form of heparan sulfate, is a linear polysaccharide with anticoagulant activity widely used in the clinic to prevent and treat thrombotic diseases. However, there are several noteworthy drawbacks associated with animal-sourced heparin during the preparation process. The in vitro enzymatic synthesis of heparin has become a promising substitute for animal-derived heparin. The synthesis of bioengineered heparin involves recombinant expression and preparation of polymerases, sulfotransferases, and an epimerase. D-glucuronyl C5-epimerase (HSepi) catalyzes D-glucuronic acids immediately adjacent to N-sulfo-glucosamine units to L-iduronic acid. Preparation of recombinant HSepi with high activity and production yield for in vitro heparin synthesis has not been resolved as of now. The findings of this study indicate that the catalytic activity of HSepi is regulated using post-translational modifications, including N-linked glycosylation and disulfide bond formation. Further mutation studies suggest that tyrosine residues, such as Tyr168, Tyr222, Tyr500, Tyr560, and Tyr578, are crucial in maintaining HSepi activity. A high-yield expression strategy was established using the lentiviral-based transduction system to produce recombinant HSepi (HSepi589) with a specific activity of up to 1.6 IU/mg. Together, this study contributes to the preparation of highly active HSepi for the enzymatic synthesis of heparins by providing additional insights into the catalytic activity of HSepi.

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Hematopoietic stem cell (HSC) gene therapy has the potential to cure many genetic, malignant, and infectious diseases. We have shown in a nonhuman primate gene therapy and transplantation model that the CD34+CD90+ cell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. In this study, we show the translational potential of this HSC-enriched CD34 subset for lentivirus-mediated gene therapy. Alternative HSC enrichment strategies include the purification of CD133+ cells or CD38low/- subsets of CD34+ cells from human blood products. We directly compared these strategies to the isolation of CD90+ cells using a good manufacturing practice (GMP) grade flow-sorting protocol with clinical applicability. We show that CD90+ cell selection results in about 30-fold fewer target cells in comparison to CD133+ or CD38low/- CD34+ hematopoietic stem and progenitor cell (HSPC) subsets without compromising the engraftment potential in vivo. Single-cell RNA sequencing confirmed nearly complete depletion of lineage-committed progenitor cells in CD90+ fractions compared to alternative selections. Importantly, lentiviral transduction efficiency in purified CD90+ cells resulted in up to 3-fold higher levels of engrafted gene-modified blood cells. These studies should have important implications for the manufacturing of patient-specific HSC gene therapy and gene-engineered cell products.

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Organoid culture faithfully reproduces the in vivo characteristics of the intestinal/colon epithelium and elucidates molecular mechanisms underlying the regulation of stem cell compartment that, if altered, may lead tumorigenesis. CRISPR-Cas9 based editing technology has provided promising opportunities for targeted loss-of-function mutations at chosen sites in the genome of eukaryotes. Herein, we demonstrate a CRISPR/Cas9-mediated mutagenesis-based screening method using murine intestinal organoids by investigating the phenotypical morphology of Cas9-expressing murine intestinal organoids. Murine intestinal crypts can be isolated and seeded into Matrigel and grown into stable organoid lines. Organoids subsequently transduced and selected to generate Cas9 expressing organoids. These organoids can be further transduced with the second lentiviruses expressing guide RNA (gRNA) (s) and screened for 8-10 days using bright-field and fluorescent microscopy to determine possible morphological or phenotypical abnormalities. Via phenotypical screening analysis, the candidate knockouts can be selected based on differential abnormal growth pattern vs their untransduced or lenti-GFP transduced controls. Further assessment of these knockout organoids can be done via phalloidin and propidium iodide (PI) staining, proliferation assay and qRT-PCR and also biochemical analysis. This CRISPR/Cas9 organoid mutagenesis-based screening method provides a reliable and rapid approach for investigating large numbers of genes with unknown/poorly identified biological functions. Knockout intestinal organoids can be associated with the key biological function of the gene(s) in development, homeostasis, disease progression, tumorigenesis, and drug screening, thereby reducing and potentially replacing animal models.

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Lentiviruses are quite effective gene delivery systems for stable production of genetically engineered human cells. However, prior to using lentivirus to deliver genetic materials to cells of interest, the normal course of production of these lentiviruses involves a lengthy collection, purification, preservation, and quantification process. In this report, we demonstrate the ability for producer HEK293T cells to simultaneously produce lentiviral particles and transduce (i.e., infect) target cells through a membrane-based coculture system in a continuous, real-time mode which negates the need for a separate viral collection and quantification process. The coculture system was evaluated for major design features such as variations in HEK293T seeding density, target cell type densities, as well as membrane porosities to identify key relationships between lentiviral particle production rate and infection kinetics for adherent and suspension cell types. As a proof-of-concept for the creation of an engineered cell immunotherapy, we describe the ability to engineer human T cells isolated from PBMCs under the control of this coculture system in under 6 days with a GFP construct. These studies suggest the capability to combine and more closely automate the transfection/transduction process in order to facilitate well-timed and cost-effective transduction of target cell types. These experiments provide novel insight into the forthcoming transition into improved manufacturing systems for viral production and subsequent cell engineering.

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PURPOSE: A major question remaining in approaches to tissue engineering and organ replacement is the role of native mobilized native cells in the regeneration process of damaged tissues and organs. The goal of this study was to compare the cell mobilizing effects of the chemokine CXCL12 and cell therapy on the urinary sphincter of nonhuman primates (NHP) with chronic intrinsic urinary sphincter dysfunction. METHODS: Either autologous lenti-M-cherry labeled skeletal muscle precursor cells (skMPCs) or CXCL12 were injected directly into the sphincter complex of female NHPs with or without surgery-induced chronic urinary sphincter dysfunction (n=4/treatment condition). All monkeys had partial bone marrow transplantation with autologous lenti-green fluorescent protein (GFP) bone marrow cells prior to treatment. Labeled cells were identified, characterized and quantified using computer-assisted immunohistochemistry 6 months posttreatment. RESULTS: GFP-labeled bone marrow cells (BMCs) were identified in the bone marrow and both BMCs and skMPCs were found in the urinary sphincter at 6-month postinjection. BMCs and skMPCs were present in the striated muscle, smooth muscle, and lamina propria/urothelium of the sphincter tissue. Sphincter injury increased the sphincter content of BMCs when analyzed 6-month postinjection. CXCL12 treatment, but not skMPCs, increased the number of BMCs in all layers of the sphincter complex (P<0.05). CXCL12 only modestly (P=0.15) increased the number of skMPCs in the sphincter complex. CONCLUSION: This dual labeling methodology now provides us with the tools to measure the relative number of locally injected cells versus bone marrow transplanted cells. The results of this study suggest that CXCL12 promotes mobilization of cells to the sphincter, which may contribute more to sphincter regeneration than injected cells.

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PURPOSE: A major question remaining in approaches to tissue engineering and organ replacement is the role of native mobilized native cells in the regeneration process of damaged tissues and organs. The goal of this study was to compare the cell mobilizing effects of the chemokine CXCL12 and cell therapy on the urinary sphincter of nonhuman primates (NHP) with chronic intrinsic urinary sphincter dysfunction. METHODS: Either autologous lenti-M-cherry labeled skeletal muscle precursor cells (skMPCs) or CXCL12 were injected directly into the sphincter complex of female NHPs with or without surgery-induced chronic urinary sphincter dysfunction (n=4/treatment condition). All monkeys had partial bone marrow transplantation with autologous lenti-green fluorescent protein (GFP) bone marrow cells prior to treatment. Labeled cells were identified, characterized and quantified using computer-assisted immunohistochemistry 6 months posttreatment. RESULTS: GFP-labeled bone marrow cells (BMCs) were identified in the bone marrow and both BMCs and skMPCs were found in the urinary sphincter at 6-month postinjection. BMCs and skMPCs were present in the striated muscle, smooth muscle, and lamina propria/urothelium of the sphincter tissue. Sphincter injury increased the sphincter content of BMCs when analyzed 6-month postinjection. CXCL12 treatment, but not skMPCs, increased the number of BMCs in all layers of the sphincter complex (P < 0.05). CXCL12 only modestly (P=0.15) increased the number of skMPCs in the sphincter complex. CONCLUSIONS: This dual labeling methodology now provides us with the tools to measure the relative number of locally injected cells versus bone marrow transplanted cells. The results of this study suggest that CXCL12 promotes mobilization of cells to the sphincter, which may contribute more to sphincter regeneration than injected cells.

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Schmallenberg virus (SBV) is a Culicoides-transmitted orthobunyavirus that poses a threat to susceptible livestock species such as cattle, sheep and goats. The nucleocapsid (N) protein of SBV is an ideal diagnostic antigen for the detection of viral infection. In this study, a stable Vero cell line, Vero-EGFP-SBV-N, constitutively expressing the SBV-N protein was established using a lentivirus system combined with puromycin selection. This cell line spontaneously emitted green fluorescent signals distributed throughout the cytoplasm, in which the expression of SBV-N fusion protein was confirmed by western blot analysis. The expression of SBV-N protein in Vero-EGFP-SBV-N cells was stable for more than fifty passages without puromycin pressure. The SBV-N fusion protein contained both an N-terminal enhanced green fluorescent protein (EGFP) tag and a C-terminal hexa-histidine (6 × His) tag, by which the N protein was successfully purified using Ni-NTA affinity chromatography. The cell line was further demonstrated to be reactive with SBV antisera and an anti-SBV monoclonal antibody in indirect immunofluorescence assays. Taken together, our results demonstrate that the Vero-EGFP-SBV-N cell line has potential for application in the serological diagnosis of SBV infection.

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The CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the α5, α3, and ß4 nicotinic acetylcholine receptor (nAChR) subunits, has been linked to nicotine dependence. The habenulo-interpeduncular (Hb-IPN) tract is particularly enriched in α3ß4 nAChRs. We recently showed that modulation of these receptors in the medial habenula (MHb) in mice altered nicotine consumption. Given that ß4 is rate-limiting for receptor activity and that single nucleotide polymorphisms (SNPs) in CHRNB4 have been linked to altered risk of nicotine dependence in humans, we were interested in determining the contribution of allelic variants of ß4 to nicotine receptor activity in the MHb. We screened for missense SNPs that had allele frequencies >0.0005 and introduced the corresponding substitutions in Chrnb4. Fourteen variants were analyzed by co-expression with α3. We found that ß4A90I and ß4T374I variants, previously shown to associate with reduced risk of smoking, and an additional variant ß4D447Y, significantly increased nicotine-evoked current amplitudes, while ß4R348C, the mutation most frequently encountered in sporadic amyotrophic lateral sclerosis (sALS), showed reduced nicotine currents. We employed lentiviruses to express ß4 or ß4 variants in the MHb. Immunoprecipitation studies confirmed that ß4 lentiviral-mediated expression leads to specific upregulation of α3ß4 but not ß2 nAChRs in the Mhb. Mice injected with the ß4-containing virus showed pronounced aversion to nicotine as previously observed in transgenic Tabac mice overexpressing Chrnb4 at endogenous sites including the MHb. Habenular expression of the ß4 gain-of-function allele T374I also resulted in strong aversion, while transduction with the ß4 loss-of function allele R348C failed to induce nicotine aversion. Altogether, these data confirm the critical role of habenular ß4 in nicotine consumption, and identify specific SNPs in CHRNB4 that modify nicotine-elicited currents and alter nicotine consumption in mice.