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Background Cell reprogramming is a promising avenue for cell-based therapies as it allows for the generation of multipotent, unipotent, or mature somatic cells without going through a pluripotent state. While the use of autologous cells is considered ideal, key challenges for their clinical translation include the ability to reproducibly generate sufficient quantities of cells within a therapeutically relevant time window. Methods We performed transfection of three distinct human somatic starting populations of cells with a non-integrating synthetic plasmid expressing Musashi 1 (MSI1), Neurogenin 2 (NGN2), and Methyl-CpG-Binding Domain 2 (MBD2). The resulting directly reprogrammed neural precursor cells (drNPCs) were examined in vitro using RT-qPCR, karyotype analysis, immunohistochemistry, and FACS at early and late time post-transfection. Electrophysiology (patch clamp) was performed on drNPC-derived neurons to determine their capacity to generate action potentials. In vivo characterization was performed following transplantation of drNPCs into two animal models (Shiverer and SCID/Beige mice), and the numbers, location, and differentiation profile of the transplanted cells were examined using immunohistochemistry. Results Human somatic cells can be directly reprogrammed within two weeks to neural precursor cells (drNPCs) by transient exposure to Msi1, Ngn2, and MBD2 using non-viral constructs. The drNPCs generate all three neural cell types (astrocytes, oligodendrocytes, and neurons) and can be passaged in vitro to generate large numbers of cells within four weeks. drNPCs can respond to in vivo differentiation and migration cues as demonstrated by their migration to the olfactory bulb and contribution to neurogenesis in vivo. Differentiation profiles of transplanted cells onto the corpus callosum of myelin-deficient mice reveal the production of oligodendrocytes and astrocytes. Conclusions Human drNPCs can be efficiently and rapidly produced from donor somatic cells and possess all the important characteristics of native neural multipotent cells including differentiation into neurons, astrocytes, and oligodendrocytes, and in vivo neurogenesis and myelination. Electronic supplementary material The online version of this article (10.1186/s13287-019-1255-4) contains supplementary material, which is available to authorized users.

Figure S7. drNPCs present in olfactory bulb express markers of neuronal differentiation. drNPCs transplanted into the SCID/Beige animals are present in the olfactory bulb at 1 month post-transplant and contain a subpopulation of NCAM-positive cells (white arrows). Scale bars = 10 μm. (PDF 831 kb)

Figure S3. drNPC differentiation in vitro. Differentiated drNPCs express increased levels of Map2 and Gfap mRNA compared to control drNPCs that were cultured in maintenance media. There was no change in Olig1 expression. Data are shown as mean ± SEM. n = 3 biological samples per cohort. Gene expression levels are relative to control drNPCs and normalized to the reference gene Gapdh. (PDF 111 kb)

Figure S8. Pixel colocalization analysis of cytoplasmic immunofluorescent staining. A: Co-localization measurement of cytoplasmic immunofluorescent staining in mouse brain cryosections. Zeiss Zen software was used to measure channel intensities of each immunofluorescently stained pixel and quantify a double positive pixel ratio in defined regions of interest. Threshold pixel intensities were adjusted first to non-labeled areas (Box 1), followed by fluorophore labeled secondary antibody backgrounds (Box 2) in the same section. Pixels of human cytoplasmic epitope-specific STEM121-labeled area (Box 3) was compared to STEM121-negative areas. The sample image, scatter plot of all three regions (Boxes 1, 2, and 3), and pixel measurements/co-localization coefficient are shown. (PDF 371 kb)

Figure S2. The synthetic plasmid used in reprogramming does not integrate into drNPCs. RT-qPCR analysis demonstrates the lack of any plasmid sequences in reprogrammed drNPCs. 4 different primer pairs were designed to detect plasmid sequence (PCRs 1-4) and 1 ubiquitous promotor primer pair was used as positive control. Samples studied were control template (determined to be ~â 10 copy numbers), untransfected BMCs, and 3 distinct samples of drNPCs (drNPC1-3) made from different starting cells. UDâ =â undetected. (PDF 137 kb)

Figure S1. drNPCs can be generated from multiple starting cell populations. In addition to BMCs, drNPCs can be generated from other cell sources such human foreskin fibroblasts (HFF) and keratinocytes. The expression of neural markers Nestin, Sox2, and GFAP is only observed post-transfection (post-reprogramming). Scale bar = 100¾m. (PDF 365 kb)

Figure S6. drNPC-derived spheres can be derived from monolayers and give rise to neurons and glia in vitro. Dissociating 80% confluent drNPC monolayers and plating them at 10 cells/μl density results in free-floating spheres within 7 days of culturing. Differentiating spheres for additional 7 days in the presence of serum will result in GFAP-positive astrocytes, Olig2-positive oligodendrocytes, and Tuj1-positive neurons. Scale bar = 100 μm. Representative bright field images of drNPC monolayer (left) and spheres (centre), and a representative IHC image of differentiated drNPC-derived spheres. (PDF 249 kb)

Figure S4. BMCs do not acquire NPC characteristic when placed in the neural stem cell culturing conditions in the absence of reprogramming. BMCs placed in the neural stem cell culturing conditions maintain expression of BMC-specific marker CD44 and Stro1 and do not acquire any NPC marker expression (Nestin, Pax6, Sox2). Early = 1–3 in vitro; mid = days 6–7 in vitro; late = days 14–16 in vitro. Nuclei stained with Hoechst (blue). Scale bar = 100 μm. (PDF 496 kb)

Little mechanical data is available on human arteries because of the difficulty of testing artery samples often obtained from autopsy, while arteries are still considered “fresh”. Various solutions mimicking the physiological environment have been used to preserve artery samples from harvesting to testing. Cryopreservation might provide a means to preserve the mechanical properties of arteries for days or weeks after harvesting. The objective of this study is to investigate the effect of several preservation methods, including simplified cryopreservation methods, on the passive mechanical properties of arteries. Eighteen fresh cruciform samples were mechanically tested. Samples were divided in three groups based on preservation medium and freezing method: isotonic saline solution, Krebs-Henseleit buffer solution with dimethyl sulfoxide (DMSO), and dipped in liquid nitrogen. In each group, half of the samples were stored at -20? and the other half at -80?. Two months later, all the tissues were thawed at 4? and mechanical tests were repeated. Preservation of arteries for two months in Krebs solution with DMSO (at -20? or at -80?) or in isotonic saline solution at -20? were the methods that least changed the mechanical properties of the arteries.

Biomaterials capable of efficient gene delivery by embedded cells provide a fundamental tool for the treatment of acquired or hereditary diseases. A major obstacle is maintaining adequate nutrient and oxygen diffusion to cells within the biomaterial. In this study, we combined the solid free-form fabrication and porogen leaching techniques to fabricate three-dimensional scaffolds, with bimodal pore size distribution, for cell-based gene delivery. The objective of this study was to design micro-/macroporous scaffolds to improve cell viability and drug delivery. Murine bone marrow-derived mesenchymal stromal cells (MSCs) genetically engineered to secrete erythropoietin (EPO) were seeded onto poly-L-lactide (PLLA) scaffolds with different microporosities. Over a period of 2 weeks in culture, an increase in cell proliferation and metabolic activity was observed with increasing scaffold microporosity. The concentration of EPO detected in supernatants also increased with increasing microporosity level. Our study shows that these constructs can promote cell viability and release of therapeutic proteins, and clearly demonstrates their capacity for a dual role as scaffolds for tissue regeneration and as delivery systems for soluble gene products.

Neural stem cells (NSCs) provide promising therapeutic potential for cell replacement therapy in spinal cord injury (SCI). However, high increases of cell viability and poor control of cell differentiation remain major obstacles. In this study, we have developed a non-woven material made of co-electrospun fibers of poly L-lactic acid and gelatin with a degradation rate and mechanical properties similar to peripheral nerve tissue and investigated their effect on cell survival and differentiation into motor neuronal lineages through the controlled release of retinoic acid (RA) and purmorphamine. Engineered Neural Stem-Like Cells (NSLCs) seeded on these fibers, with and without the instructive cues, differentiated into β-III-tubulin, HB-9, Islet-1, and choactase-positive motor neurons by immunostaining, in response to the release of the biomolecules. In addition, the bioactive material not only enhanced the differentiation into motor neuronal lineages but also promoted neurite outgrowth. This study elucidated that a combination of electrospun fiber scaffolds, neural stem cells, and controlled delivery of instructive cues could lead to the development of a better strategy for peripheral nerve injury repair.

Gene therapy for hemophilia B and other hereditary plasma protein deficiencies showed great promise in pre-clinical and early clinical trials. However, safety concerns about in vivo delivery of viral vectors and poor post-transplant survival of ex vivo modified cells remain key hurdles for clinical translation of gene therapy. We here describe a 3D scaffold system based on porous hydroxyapatite-PLGA composites coated with biomineralized collagen 1. When combined with autologous gene-engineered factor IX (hFIX) positive mesenchymal stem cells (MSCs) and implanted in hemophilic mice, these scaffolds supported long-term engraftment and systemic protein delivery by MSCs in vivo. Optimization of the scaffolds at the macro-, micro- and nanoscales provided efficient cell delivery capacity, MSC self-renewal and osteogenesis respectively, concurrent with sustained delivery of hFIX. In conclusion, the use of gene-enhanced MSC-seeded scaffolds may be of practical use for treatment of hemophilia B and other plasma protein deficiencies. © 2010 Elsevier Ltd.

Engineered scaffolds for tissue-engineering should be designed to match the stiffness and strength of healthy tissues while maintaining an interconnected pore network and a reasonable porosity. In this work, we have used 3D-plotting technique to produce poly-L-Lactide macroporous scaffolds with two different pore sizes. The ability of these macroporous scaffolds to support chondrocyte attachment and viability were compared under static and dynamic loading in vitro. Moreover, the 3D-plotting technique was combined with porogen-leaching, leading to macro/microporous scaffolds, so as to examine the effect of microporosity on the level of cell attachment and viability under similar loading condition.Canine chondrocytes’ cells were seeded onto the scaffolds with different topologies, and the constructs were cultured for up to 2 weeks under static conditions or in a bioreactor under dynamic compressive strain of 10% strain, at a frequency of 1 Hz. The attachment and cell growth of chondrocytes were examined by scanning electron microscopy and by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. A significant difference in cell attachment was observed in macroporous scaffolds with different pore sizes after 1, 7, and 14 days. Cell viability in the scaffolds was enhanced with decreasing pore size and increasing microporosity level throughout the culture period. Chondrocyte viability in the scaffolds cultured under dynamic loading was significantly higher (p

In an effort to design biomaterials that may promote repair of the central nervous system, 3-dimensional scaffolds made of electrospun poly lactic acid nanofibers with interconnected pores were fabricated. These scaffolds were functionalized with polyallylamine to introduce amine groups by wet chemistry. Experimental conditions of the amination protocol were thoroughly studied and selected to introduce a high amount of amine group while preserving the mechanical and structural properties of the scaffold. Subsequent covalent grafting of epidermal growth factor was then performed to further tailor these aminated structures. The scaffolds were then tested for their ability to support Neural Stem-Like Cells (NSLCs) culture. Of interest, NSLCs were able to proliferate on these EGF-grafted substrates and remained viable up to 14 d even in the absence of soluble growth factors in the medium.

We have previously shown that acute intravenous injections of moxonidine and clonidine increase plasma atrial natriuretic peptide (ANP), a vasodilator, diuretic and natriuretic hormone. We hypothesized that moxonidine stimulates the release of ANP, which would act on its renal receptors to cause diuresis and natriuresis, and these effects may be altered in hypertension. Moxonidine (0, 10, 50, 100 or 150 microg in 300 microl saline) and clonidine (0, 1, 5 or 10 microg in 300 microl saline) injected intravenously in conscious normally hydrated normotensive Sprague-Dawley rats (SD, approximately 200 g) and 12-14-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) dose-dependently stimulated diuresis, natriuresis, kaliuresis and cGMP excretion, with these effects being more pronounced during the first hour post-injection. The actions of 5 microg clonidine and 50 microg moxonidine were inhibited by yohimbine, an alpha2-adrenoceptor antagonist, and efaroxan, an imidazoline I1-receptor antagonist. Moxonidine (100 microg) stimulated (P0.01) diuresis in SHR (0.21+/-0.04 vs 1.16+/-0.06 ml h(-1) 100 g(-1)), SD (0.42+/-0.06 vs 1.56+/-0.19 ml h(-1) 100 g(-1)) and WKY (0.12+/-0.04 vs 1.44+/-0.21 ml h(-1) 100 g(-1)). Moxonidine-stimulated urine output was lower in SHR than in SD and WKY. Moxonidine-stimulated sodium and potassium excretions were lower in SHR than in SD, but not WKY, demonstrating an influence of strain but not of pressure. Pretreatment with the natriuretic peptide antagonist anantin (5 or 10 microg) resulted in dose-dependent inhibition of moxonidine-stimulated urinary actions. Anantin (10 microg) inhibited (P0.01) urine output to 0.38+/-0.06, 0.12+/-0.01, and 0.16+/-0.04 ml h(-1) 100 g(-1) in SD, WKY, and SHR, respectively. Moxonidine increased (P0.01) plasma ANP in SD (417+/-58 vs 1021+/-112 pg ml(-1)) and WKY (309+/-59 vs 1433+/-187 pg ml(-1)), and in SHR (853+/-96 vs 1879+/-229 pg ml(-1)). These results demonstrate that natriuretic peptides mediate the urinary actions of moxonidine through natriuretic peptide receptors.

Acute intravenous administration of moxonidine, an imidazoline I1-receptor agonist, reduces blood pressure (BP) in normotensive and hypertensive rats, induces diuresis and natriuresis, and stimulates plasma atrial natriuretic peptide (ANP). In these studies we investigated the involvement of natriuretic peptides (ANP and brain natriuretic peptide) in the effects of chronic activation of imidazoline receptors.Spontaneously hypertensive rats (SHR; 12 to 14 weeks old) received 7-day moxonidine treatment at various doses (10, 20, 60, and 120 microg/kg/h) via subcutaneously implanted osmotic minipumps.Hemodynamic parameters (continuously monitored by telemetry) revealed that, compared with saline-treated rats, moxonidine dose-dependently decreased blood pressures (BPs). Maximal blood pressure lowering effect was achieved by day 4 of treatment, at which point 60 microg/kg/h reduced mean arterial pressure (MAP) by 14.5 +/- 6.8 mm Hg as compared with basal levels. The decrease in MAP was influenced by a drop in both diastolic and systolic pressures. Moxonidine treatment did not alter daily urinary sodium and potassium excretions, but 120 microg/kg/h moxonidine decreased urine volume after 2 days and increased cyclic guanosine 3'5'monophosphate excretion on days 4 to 7 of treatment. Chronic moxonidine treatment dose-dependently increased plasma ANP to reach, at 120 microg/kg/h, a 40% increase (P.01) above that of corresponding saline-treated SHR, with a concomitant increase in left and right atrial ANP mRNA (more than twofold). Plasma BNP increased by 120 microg/kg/h moxonidine (11.0 +/- 1.1 v 16.5 +/- 1.9 pg/mL, P.002) without significant increases in atrial and ventricular BNP mRNA.ANP and BNP may be involved in the antihypertensive effect of chronic moxonidine treatment. Accordingly, natriuretic peptides may contribute to the sympatholytic and cardioprotective effects of chronic activation of imidazoline I1-receptors.

Figure S5. Pluripotency markers are not expressed during reprogramming. BMCs reprogrammed to drNPCs were analyzed for pluripotency markers Nanog, Oct4, SSEA-1, and TRA1–81 during the reprogramming process over time. No expression of pluripotency markers was observed. Early = 1–3 in vitro; mid = days 6–7 in vitro; late = days 14–16 in vitro. Nuclei stained with Hoechst (blue). Scale bar = 100 μm. (PDF 562 kb)

Figure S5. Pluripotency markers are not expressed during reprogramming. BMCs reprogrammed to drNPCs were analyzed for pluripotency markers Nanog, Oct4, SSEA-1, and TRA1–81 during the reprogramming process over time. No expression of pluripotency markers was observed. Early = 1–3 in vitro; mid = days 6–7 in vitro; late = days 14–16 in vitro. Nuclei stained with Hoechst (blue). Scale bar = 100 μm. (PDF 562 kb)