Unfolded protein response is required for the definitive endodermal specification of mouse embryonic stem cells via Smad2 and ß-catenin signaling.

Tremendous efforts have been made to elucidate the molecular mechanisms that control the specification of definitive endoderm cell fate in gene knockout mouse models and ES cell (ESC) differentiation models. However, the impact of the unfolded protein response (UPR), because of the stress of the endoplasmic reticulum on endodermal specification, is not well addressed. We employed UPR-inducing agents, thapsigargin and tunicamycin, in vitro to induce endodermal differentiation of mouse ESCs. Apart from the endodermal specification of ESCs, Western blotting demonstrated the enhanced phosphorylation of Smad2 and nuclear translocation of ß-catenin in ESC-derived cells. The inclusion of the endoplasmic reticulum stress inhibitor tauroursodeoxycholic acid to the induction cultures prevented the differentiation of ESCs into definitive endodermal cells even when Activin A was supplemented. Also, the addition of the TGF-ß inhibitor SB431542 and the Wnt/ß-catenin antagonist IWP-2 negated the endodermal differentiation of ESCs mediated by thapsigargin and tunicamycin. These data suggest that the activation of the UPR appears to orchestrate the induction of the definitive endodermal cell fate of ESCs via both the Smad2 and ß-catenin signaling pathways. The prospective regulatory machinery may be helpful for directing ESCs to differentiate into definitive endodermal cells for cellular therapy in the future.

Lentivirus-modified hematopoietic stem cell gene therapy for advanced symptomatic juvenile metachromatic leukodystrophy: A long-term follow-up pilot study.

Metachromatic leukodystrophy (MLD) is an inherited disease caused by a deficiency of the enzyme arylsulfatase A (ARSA). Lentivirus-modified autologous hematopoietic stem cell gene therapy (HSCGT) has recently been approved for clinical use in pre- and early-symptomatic children with MLD to increase ARSA activity. Unfortunately, this advanced therapy is not available for most patients with MLD who have progressed to more advanced symptomatic stages at diagnosis. Patients with late-onset juvenile MLD typically present with a slower neurological progression of symptoms and represent a significant burden to the economy and healthcare system, whereas those with early-onset infantile MLD die within a few years of symptom onset. We conducted a pilot study to determine the safety and benefit of HSCGT in patients with post-symptomatic juvenile MLD and report preliminary results. The safety profile of HSCGT was favorable in this long-term follow-up over nine years. The most common adverse events (AEs) within two months of HSCGT were related to busulfan conditioning, and all AEs resolved. No HSCGT-related AEs and no evidence of distorted hematopoietic differentiation during long-term follow-up for up to 9.6 years. Importantly, to date, patients have maintained remarkably improved ARSA activity with a stable disease state, including increased Functional Independence Measure (FIM) score and decreased magnetic resonance imaging (MRI) lesion score. This long-term follow-up pilot study suggests that HSCGT is safe and provides clinical benefit to patients with post-symptomatic juvenile MLD.

The tetraspanin CD9 regulates migration, adhesion, and homing of human cord blood CD34+ hematopoietic stem and progenitor cells.

The stromal cell-derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis plays a critical role in homing and engraftment of hematopoietic stem/progenitor cells (HSCs) during bone marrow transplantation. To investigate the transcriptional regulation provided by this axis, we performed the first differential transcriptome profiling of human cord blood CD34(+) cells in response to short-term exposure to SDF-1 and identified a panel of genes with putative homing functions. We demonstrated that CD9, a member of the tetraspanin family of proteins, was expressed in CD34(+)CD38(-/lo) and CD34(+)CD38(+) cells. CD9 levels were enhanced by SDF-1, which simultaneously down-regulated CXCR4 membrane expression. Using specific inhibitors and activators, we demonstrated that CD9 expression was modulated via CXCR4, G-protein, protein kinase C, phospholipase C, extracellular signal-regulated kinase, and Janus kinase 2 signals. Pretreatment of CD34(+) cells with the anti-CD9 monoclonal antibody ALB6 significantly inhibited SDF-1-mediated transendothelial migration and calcium mobilization, whereas adhesion to fibronectin and endothelial cells was enhanced. Pretreatment of CD34(+) cells with ALB6 significantly impaired their homing to bone marrow and spleen of sublethally irradiated NOD/SCID (nonobese diabetic/severe combined immune-deficient) mice. Sorted CD34(+)CD9(-) cells displayed lower bone marrow homing capacity compared with that of total CD34(+) cells. CD9 expression on homed CD34(+) cells was significantly up-regulated in vivo. Our results indicate that CD9 might possess specific functions in HSC homing.

DNA microarray expression analysis of baicalin-induced differentiation of C17.2 neural stem cells.

Identifying baicalin-regulated genes for neuronal differentiation: Baicalin is a potent neuronal-differentiation-inducing compound. This study explored the gene expression regulated through baicalin-induced differentiation of C17.2 neural stem cells by using a DNA microarray followed by qPCR validation. The expression of 15 genes was significantly regulated among the 58 differentially expressed genes important for nervous system development and function.

Quantitative assessment of gait and neurochemical correlation in a classical murine model of Parkinson's disease.

BACKGROUND: Gait deficits are important clinical symptoms of Parkinson's disease (PD). However, existing behavioral tests for the detection of motor impairments in rodents with systemic dopamine depletion only measure akinesia and dyskinesia, and data focusing on gait are scarce. We evaluated gait changes in the methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced C57BL/6 murine model of PD by using a computer-assisted CatWalk system. Correlations of gait parameters with tyrosine hydroxylase (TH) protein levels in the substantia nigra (SN) were also investigated. RESULTS: The gait readouts, including the walking duration, variation of walking speed, step cycle, duty cycle, stance, initial dual stance, terminal dual stance, three- and four-point supports, and the base of support between hind limbs was noted to increase significantly one week after MPTP injection. In contrast, values of the stride length, cadence, swing speed, and diagonal dual support decreased substantially following MPTP treatment (p < 0.05). All of these changes lasted for three weeks after the last MPTP administration. Except for the stance in the fore limbs and the swing speed in the hind limbs, the gait variability in the PD mice showed a closer correlation with the protein levels of TH in the SN than the walking distances in the conventional open field test. Coordination parameters of the regularity index and step pattern were not affected in mice treated with MPTP. CONCLUSION: Data of the study suggest that the computer-assisted CatWalk system can provide reliable and objective criteria to stratify gait changes arising from MPTP-induced bilateral lesions in C57/BL6 mice. The extent of gait changes was noted to correlate with the expression of the biomarker for dopaminergic neurons. This novel analytical method may hold promise in the study of disease progression and new drug screening in a murine PD model.

High cystic fibrosis transmembrane conductance regulator expression in childhood B-cell acute lymphoblastic leukemia acts as a potential therapeutic target.

Background: The role of cystic fibrosis transmembrane conductance regulator (CFTR) in hematopoiesis and adult leukemia has been demonstrated using a zebrafish model and leukemia cell lines in our previous works. Here, we continue to explore the association between CFTR and human childhood B-cell acute lymphoblastic leukemia (B-ALL). Methods: We continued to collect the peripheral blood and bone marrows of human childhood patients diagnosed with primary B-ALL as well as non-leukemia controls and isolated lymphocytes for analysis using western blotting and quantitative real-time polymerase chain reaction (qPCR) assay. Then, we used immunofluorescence, co-immunoprecipitation, western blotting, luciferase, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assays to identify the interaction of CFTR with Wnt signaling in B-ALL. Finally, we established B-ALL xenograft model in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice using SUP-B15 cells, and examined whether the CFTR inhibitor CFTR-inh172 could active against SUP-B15-Dependent B-ALL in vivo. Results: Highly expressed CFTR protein and mRNA are associated with primary childhood B-ALL patients. Aberrantly upregulated CFTR and Wnt signaling, our previously reported CFTR-Dvl2-ß-catenin pathway, is found in human childhood B-ALL patients. Interference with CFTR in B-ALL cell lines induces the downregulation of DVL2/ß-catenin and Wnt downstream target accompanied by a reduction of cell proliferation. Furthermore, B-ALL cell lines SUP-B15 cell-transplanted NOD/SCID mice treated with CFTR inhibitor CFTRinh-172 had significantly longer survival and slower leukemia progression compared with mice treated with vehicle dimethyl sulfoxide (DMSO). Conclusions: These findings demonstrate that highly expressed CFTR is associated with human childhood B-ALL and the potential of CFTR inhibitor CFTR-inh172 for the treatment of human B-ALL.

Neuronal differentiation of C17.2 neural stem cells induced by a natural flavonoid, baicalin.

Natural medicinal materials are a significant resource for the identification of compounds with specific biological properties. In this study, we employed multipotent C17.2 neural stem cells as a model for screening natural compounds that possess neural differentiation-inducing properties. We show here for the first time that, out of the 67 compounds tested, the flavonoid baicalin is a potent differentiation-inducing agent. Baicalin increased the number of cells bearing extended neurites and the expression levels of a number of neuronal markers. Importantly, baicalin promoted the expression of several key neurogenic transcriptional factors. Moreover, we demonstrated that baicalin enhanced the phosphorylation/activation of Erk1/2. Inhibition of Erk1/2 activation by the MEK inhibitor U0126 attenuated the neuronal differentiation-inducing effect of baicalin. Taken together, our findings suggest that baicalin induces neuronal differentiation of C17.2 neural stem cells and that this is mediated by activation of Erk1/2. Our work lays the foundation for exploring baicalin for the promotion of neural regeneration after injury or disease.

Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow, umbilical cord and placenta: implication in the migration.

Umbilical cord (UC) and placenta (P) have been suggested as alternatives to bone marrow (BM) as sources of mesenchymal stem cells (MSC) for cell therapy, with both UC- and P-MSC possess immunophenotypic and functional characteristics similar to BM-MSC. However, under defined conditions, the migration capacity of BM- and P-MSC was found to be 5.9- and 3.2-folds higher than that of UC-MSC, respectively. By the use of 2-DE and combined MS and MS/MS analysis, six differentially expressed proteins were identified among these MSC samples, with five of them known to be involved in cell migration as migration enhancing or inhibiting proteins. Interestingly, the expression levels of those proteins reflect perfectly the migration capacity of corresponding MSC, which is also proved by in vitro overexpression and silencing techniques. Our study indicates that a bunch of migration-related proteins are pivotal in governing the migration capacity of MSC.

Improved survival outcome of childhood acute myeloid leukemia with intensified chemotherapy in Chinese children.

With the use of intensive chemotherapy and hematopoietic stem cell transplantation (HSCT), the prognosis of childhood acute myeloid leukemia (AML) improved over the last 2 decades. Survival data of Chinese pediatric patients were seldom reported. The authors adopted modified UK Medical Research Council (MRC) AML protocols for treatment of childhood AML since 1994. From 1994 to 2008, the outcomes of Chinese AML patients were studied. Sixty-eight patients were studied. The median age at diagnosis was 9.9 years. Twenty-five patients (36.8%) had favorable cytogenetic karyotypes, including t(15;17), t(8;21) and inv(16). Complete remission (CR) rate was 91.2%. The relapse rate was 29.4%. For non-M3 patients, the 5-year overall survival (pOS) was 64% ± 7% and event-free survival (pEFS) was 53% ± 7%. For those non-good-risk patients who achieved CR, there were no significant differences in outcomes between patients who received HSCT in CR1 and those received chemotherapy alone (5-year pOS 80% ± 13% and 69% ± 9%, P = .52), 5-year pEFS 69% ± 15% and 55% ± 10%, P = .40). The pOS of the 20 relapsed patients was 29% ± 11%. Sixteen patients with t(8;21) and inv(16) had similar outcome with those without favorable cytogenetics (pOS 66% ± 12% versus 65% ± 7%, P = .39; pEFS 60% ± 11% versus 54% ± 8%, P = .45). Patients who achieved CR after 2 or more courses of chemotherapy and presenting white blood cell count (WBC) ≥ 100 × 10(9)/L had poorer outcome (pOS 40% versus 80%P < .01; 43% versus 70%, P = .02, respectively). Intensified chemotherapy improved outcome of Chinese AML children. CR after first course of chemotherapy and WBC at diagnosis were important prognostic factors.

CD9 blockade suppresses disease progression of high-risk pediatric B-cell precursor acute lymphoblastic leukemia and enhances chemosensitivity.

CD9 has been implicated in cancer progression but its prognostic relevance and therapeutic potential in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are largely unknown. In a cohort of pediatric BCP-ALL patients, we found that CD9+ cases had a significantly lower 5-year relapse-free survival rate than CD9- cases. Multivariate analysis demonstrated that CD9 positivity independently predicted inferior survival outcomes, and could be applied with established prognostic features, including prednisone response and cytogenetic status, to refine patient stratification. Administration of CD9 antibody substantially suppressed disease progression in NOD/SCID mice xenografted with CD9+ cell lines and primary leukemic blasts from patients with high-risk and refractory BCP-ALL, without compromising hematopoietic stem cell engraftment. Combination of anti-CD9 with conventional chemotherapy further reduced leukemic burden and prolonged animal survival. Mechanistically, CD9 blockade inhibited leukemic cell proliferation, induced G0/G1 cell cycle arrest, activated p38, and enhanced chemotherapeutic agent-induced apoptosis. Further, CD9 physically interacted with integrin very late antigen-4, regulated affinity to vascular cell adhesion molecule-1, and was involved in leukemia-stroma interaction. Collectively, our study established CD9 as a new prognostic marker, validated the preclinical efficacy of CD9 antibody, and laid the foundation for clinical development of CD9-targeted therapy for high-risk and refractory pediatric BCP-ALL.

Comparative proteomic analysis of mesenchymal stem cells derived from human bone marrow, umbilical cord, and placenta: implication in the migration.

Umbilical cord (UC) and placenta (P) have been suggested as alternatives to bone marrow (BM) as sources of mesenchymal stem cells (MSC) for cell therapy, with both UC- and P-MSC possess immunophenotypic and functional characteristics similar to BM-MSC. However, their migration capacity, which is indispensable during tissue regeneration process, is unclear. Under defined conditions, the migration capacity of BM- and P-MSC was found 5.9- and 3.2-folds higher than that of UC-MSC, respectively. By the use of 2-DE and combined MS and MS/MS analysis, six differentially expressed proteins were identified among these MSC samples, with five of them known to be involved in cell migration as migration enhancing or inhibiting proteins. Consistent with their migration capacity, the levels of migration enhancing proteins including cathepsin B, cathepsin D and prohibitin,were significantly lower in UC-MSC when compared with those in BM- and P-MSC. For the migration inhibiting proteins such as plasminogen activator inhibitor-1 (PAI-1) and manganese superoxide dismutase, higher expression was found in the UC-MSC. We also showed that the overexpression of the PAI-1 impaired the migration capacity of BM- and P-MSC while silencing of PAI-1 enhanced the migration capacity of UC-MSC. Our study indicates that PAI-1 and other migration-related proteins are pivotal in governing the migration capacity of MSC.

Neurotrophism of bone marrow stromal cells to embryonic stem cells: noncontact induction and transplantation to a mouse ischemic stroke model.

Embryonic stem (ES) cell-derived cell products may serve as a source of cells for regenerative medicine. Currently available technologies for the induction of ES cells into neural lineage cells require extended culturing in vitro and complex procedural manipulations, with variable yields of heterogeneous cells, which have hindered the prospective use of cell derivatives for treatment of ischemic stroke. We established a simple and efficient method to derive mouse ES cells into neural lineage cells using an 8-day coculture with the bone marrow stromal cells MS5, followed by a 6-day propagation culture and a 4-day selection culture. The protocol generated a relatively high yield of neural lineage cells without any mesodermal and endodermal lineage commitment. In in vivo study, these derived cells could improve the cognitive function of ischemic stroke mice. Three weeks after transplantation, migration of implanted cells to lesioned areas was noted. It was also evident of a normalization of pyramidal neuron density and morphology in hippocampal CA1 region. One (1/17) episode of teratoma development was noted. Data suggested that MS5 cells may exert a neurotrophic effect to enhance neural differentiation of ES cells and MS5-induced ES cell-derived cells appeared to be applicable to cell therapy for ischemic stroke.

Therapeutic Potential of Human Amniotic Epithelial Cells on Injuries and Disorders in the Central Nervous System.

Despite recent advances in neurosurgery and pharmaceuticals, contemporary treatments are ineffective in restoring lost neurological functions in patients with injuries and disorders of the central nervous system (CNS). Therefore, novel and effective therapies are urgently needed. Recent studies have indicated that stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs), could repair/replace damaged or degenerative neurons and improve functional recovery in both preclinical and clinical trials. However, there are many unanswered questions and unsolved issues regarding stem cell therapy in terms of potency, stability, oncogenicity, immune response, cell sources, and ethics. Currently, human amniotic epithelial cells (hAECs) derived from the amnion exhibit considerable advantages over other stem cells and have drawn much attention from researchers. hAECs are readily available, pose no ethical concerns, and have little risk of tumorigenicity and immunogenicity. Mounting evidence has shown that hAECs can promote neural cell survival and regeneration, repair affected neurons, and reestablish damaged neural connections. It is suggested that hAECs may be the most promising candidate for cell-based therapy of neurological diseases. In this review, we mainly focus on recent advances and potential applications of hAECs for treating various CNS injuries and neurodegenerative disorders. We also discuss current hurdles and challenges regarding hAEC therapies.

A Distinct Subset of Highly Proliferative and Lentiviral Vector (LV)-Transducible NK Cells Define a Readily Engineered Subset for Adoptive Cellular Therapy.

Genetic engineering is an important tool for redirecting the function of various types of immune cells and their use for therapeutic purpose. Although NK cells have many beneficial therapeutic features, genetic engineering of immune cells for targeted therapy focuses mostly on T cells. One of the major obstacles for NK cell immunotherapy is the lack of an efficient method for gene transfer. Lentiviral vectors have been proven to be a safe tool for genetic engineering, however lentiviral transduction is inefficient for NK cells. We show in this study that lentiviral vectors pseudotyped with a modified baboon envelope glycoprotein can transduce NK cells 20-fold or higher in comparison to VSV-G pseudotyped lentiviral vector. When we investigated the mechanism of transduction, we found that activated NK cells expressed baboon envelope receptor ASCT-2. Further analysis revealed that only a subset of NK cells could be expanded and transduced with an expression profile of NK56bright, CD16dim, TRAILhigh, and CX3CR1neg. Using CD19-CAR, we could show that CD19 redirected NK cells efficiently and specifically kill cell lines expressing CD19. Taken together, the results from this study will be important for future genetic modification and for redirecting of NK cell function for therapeutic purpose.

Corrigendum: A Distinct Subset of Highly Proliferative and Lentiviral Vector (LV)-Transducible NK Cells Define a Readily Engineered Subset for Adoptive Cellular Therapy.

[This corrects the article DOI: 10.3389/fimmu.2019.02001.].

The plant mannose-binding lectin NTL preserves cord blood haematopoietic stem/progenitor cells in long-term culture and enhances their ex vivo expansion.

Ex vivo expansion of haematopoietic stem and progenitor cells in cytokine combinations is effective in promoting differentiation and proliferation of multilineage progenitor cells, but often results in reduction of self-renewable stem cells. This study investigated the effect of a mannose-binding lectin, NTL, purified from Narcissus tazetta var. chinensis, on prolonged maintenance and expansion of cord blood CD34+ cells. Our results showed that the presence of NTL or Flt-3 ligand (FL) significantly preserved a population of early stem/progenitor cells in a serum- and cytokine-free culture for 35 d. The effect of NTL on the ex vivo expansion of CD34+ cells in the presence of stem cell factor, thrombopoietin (TPO) and FL was also investigated. NTL-enhanced expansion of early progenitors (CD34+, CD34+CD38-, mixed colony-forming units and CFU-GEMM) and committed progenitor cells (granulocyte CFU, erythroid burst-forming units/CFU and megakayocyte CFU) after 8 and 12 d of culture. Six weeks after transplanting 12 d-expanded cells to non-obese diabetic severe combined immunodeficient mice, increased engraftment of human CD45+ cells was observed in the bone marrow of animals that received NTL-treated cells. The dual functions of NTL on long-term preservation and expansion of early stem/multilineage progenitor cells could be developed for applications in various cell therapy strategies, such as the clinical expansion of CD34+ cells for transplantation.

Implantation of neural stem cells embedded in hyaluronic acid and collagen composite conduit promotes regeneration in a rabbit facial nerve injury model.

The implantation of neural stem cells (NSCs) in artificial scaffolds for peripheral nerve injuries draws much attention. NSCs were ex-vivo expanded in hyaluronic acid (HA)-collagen composite with neurotrophin-3, and BrdU-labeled NSCs conduit was implanted onto the ends of the transected facial nerve of rabbits. Electromyography demonstrated a progressive decrease of current threshold and increase of voltage amplitude in de-innervated rabbits after implantation for one, four, eight and 12 weeks compared to readouts derived from animals prior to nerve transection. The most remarkable improvement, observed using Electrophysiology, was of de-innervated rabbits implanted with NSCs conduit as opposed to de-innervated counterparts with and without the implantation of HA-collagen, NSCs and HA-collagen, and HA-collagen and neurotrophin-3. Histological examination displayed no nerve fiber in tissue sections of de-innervated rabbits. The arrangement and S-100 immunoreactivity of nerve fibers in the tissue sections of normal rabbits and injured rabbits after implantation of NSCs scaffold for 12 weeks were similar, whereas disorderly arranged minifascicles of various sizes were noted in the other three arms. BrdU+ cells were detected at 12 weeks post-implantation. Data suggested that NSCs embedded in HA-collagen biomaterial could facilitate re-innervations of damaged facial nerve and the artificial conduit of NSCs might offer a potential treatment modality to peripheral nerve injuries.

CFTR mutation enhances Dishevelled degradation and results in impairment of Wnt-dependent hematopoiesis.

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) with a multitude of clinical manifestations. Some CF patients develop clinically significant anemia, suggesting that CFTR may regulate hematopoiesis. Here, we report that cftr mutant zebrafish model exhibits primitive and definitive hematopoietic defects with impaired Wnt signaling. Cftr is found to interact, via its PDZ-binding domain (PDZBD), with Dishevelled (Dvl), a key component of Wnt signaling required for hematopoietic progenitor specification, thus protecting Dvl from Dapper1 (Dpr1)-induced lysosomal degradation. Defective hematopoiesis and impaired Wnt signaling in cftr mutant can be rescued by overexpression of wild-type or channel function-defective G551D mutant CFTR with an intact PDZBD, but not Cftr with mutations in the PDZBD. Analysis of human database ( http://r2.amc.nl ) shows that CFTR is positively correlated with DVL2 and Wnt-related hematopoietic factors in human blood system. The results reveal a previously unrecognized role of CFTR, which is independent of its channel function, in regulating DVL degradation and thus Wnt signaling required for hematopoiesis in both zebrafish and humans, providing an explanation for the anemic phenotype of CF patients.

Modulation and impact of class I major histocompatibility complex by neural stem cell-derived neurotrophins on neuroregeneration.

Transplantation of neural stem cells (NSC) has shown to elicit functional recovery in experimental animal and human models of neural disorders pertaining to cell loss or degeneration. However, the underlying mechanisms of the regimen are not well understood. The scenarios lead to the speculation of neuroregeneration and replacement of lost neurons in both the central nervous system (CNS) and the peripheral nervous system (PNS). The repair per se is extremely complex involving the re-building and modulation of synapses, neurites, neural cells and glial cells. Neurotrophins, which nourish the CNS and the PNS, may attribute to the functional improvement after neural stem cell transplantation. Recent studies suggested the CNS plasticity may be modulated by the class I major histocompatibility complex (MHC), which are in turn regulated by neurotrophins. Based upon these findings, we speculate that the neurotrophins derived from the transplanted NSC may modulate the expression of the major histocompatibility complex in the injured microenvironment to facilitate neurological recovery. The proposition may have clues on the development of novel treatment modality to cure CNS injury.