Preclinical long-term safety of intraspinal transplantation of human dorsal spinal GABA neural progenitor cells.

Human pluripotent stem cell (hPSC)-derived neurons have shown promise in treating spinal cord injury (SCI). We previously showed that hPSC-derived dorsal spinal γ-aminobutyric acid (GABA) neurons can alleviate spasticity and promote locomotion in rats with SCI, but their long-term safety remains elusive. Here, we characterized the long-term fate and safety of human dorsal spinal GABA neural progenitor cells (NPCs) in naive rats over one year. All grafted NPCs had undergone differentiation, yielding mainly neurons and astrocytes. Fully mature human neurons grew many axons and formed numerous synapses with rat neural circuits, together with mature human astrocytes that structurally integrated into the rat spinal cord. The sensorimotor function of rats was not impaired by intraspinal transplantation, even when human neurons were activated or inhibited by designer receptors exclusively activated by designer drugs (DREADDs). These findings represent a significant step toward the clinical translation of human spinal neuron transplantation for treating SCI.

Spinal dI4 Interneuron Differentiation From Human Pluripotent Stem Cells.

Spinal interneurons (INs) form intricate local networks in the spinal cord and regulate not only the ascending and descending nerve transduction but also the central pattern generator function. They are therefore potential therapeutic targets in spinal cord injury and diseases. In this study, we devised a reproducible protocol to differentiate human pluripotent stem cells (hPSCs) from enriched spinal dI4 inhibitory GABAergic INs. The protocol is designed based on developmental principles and optimized by using small molecules to maximize its reproducibility. The protocol comprises induction of neuroepithelia, patterning of neuroepithelia to dorsal spinal progenitors, expansion of the progenitors in suspension, and finally differentiation into mature neurons. In particular, we employed both morphogen activators and inhibitors to restrict or "squeeze" the progenitor fate during the stage of neural patterning. We use retinoic acid (RA) which ventralizes cells up to the mid-dorsal region, with cyclopamine (CYC), an SHH inhibitor, to antagonize the ventralization effect of RA, yielding highly enriched dI4 progenitors (90% Ptf1a+, 90.7% Ascl1+). The ability to generate enriched spinal dI4 GABAergicINs will likely facilitate the study of human spinal IN development and regenerative therapies for traumatic injuries and diseases of the spinal cord.

Human spinal GABA neurons alleviate spasticity and improve locomotion in rats with spinal cord injury.

Spinal cord injury (SCI) often results in spasticity. There is currently no effective therapy for spasticity. Here, we describe a method to efficiently differentiate human pluripotent stem cells from spinal GABA neurons. After transplantation into the injured rat spinal cord, the DREADD (designer receptors exclusively activated by designer drug)-expressing spinal progenitors differentiate into GABA neurons, mitigating spasticity-like response of the rat hindlimbs and locomotion deficits in 3 months. Administering clozapine-N-oxide, which activates the grafted GABA neurons, further alleviates spasticity-like response, suggesting an integration of grafted GABA neurons into the local neural circuit. These results highlight the therapeutic potential of the spinal GABA neurons for SCI.

Temporal expression profiles of lncRNA and mRNA in human embryonic stem cell-derived motor neurons during differentiation.

BACKGROUND: Human embryonic stem cells (hESC) have been an invaluable research tool to study motor neuron development and disorders. However, transcriptional regulation of multiple temporal stages from ESCs to spinal motor neurons (MNs) has not yet been fully elucidated. Thus, the goals of this study were to profile the time-course expression patterns of lncRNAs during MN differentiation of ESCs and to clarify the potential mechanisms of the lncRNAs that are related to MN differentiation. METHODS: We utilized our previous protocol which can harvest motor neuron in more than 90% purity from hESCs. Then, differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) during MN differentiation were identified through RNA sequencing. Bioinformatic analyses were performed to assess potential biological functions of genes. We also performed qRT-PCR to validate the DElncRNAs and DEmRNAs. RESULTS: A total of 441 lncRNAs and 1,068 mRNAs at day 6, 443 and 1,175 at day 12, and 338 lncRNAs and 68 mRNAs at day 18 were differentially expressed compared with day 0. Bioinformatic analyses identified that several key regulatory genes including POU5F1, TDGF1, SOX17, LEFTY2 and ZSCAN10, which involved in the regulation of embryonic development. We also predicted 283 target genes of DElncRNAs, in which 6 mRNAs were differentially expressed. Significant fold changes in lncRNAs (NCAM1-AS) and mRNAs (HOXA3) were confirmed by qRT-PCR. Then, through predicted overlapped miRNA verification, we constructed a lncRNA NCAM1-AS-miRNA-HOXA3 network.

Human adipose-derived Mesenchymal stem cells, low-intensity pulsed ultrasound, or their combination for the treatment of knee osteoarthritis: study protocol for a first-in-man randomized controlled trial.

BACKGROUND: Human adipose-derived Mesenchymal stem cells (HADMSCs) have proven their efficacy in treating osteoarthritis (OA), in earlier preclinical and clinical studies. As the tissue repairers are under the control of mechanical and biochemical signals, improving regeneration outcomes using such signals has of late been the focus of attention. Among mechanical stimuli, low-intensity pulsed ultrasound (LIPUS) has recently shown promise both in vitro and in vivo. This study will investigate the potential of LIPUS in enhancing the regeneration process of an osteoarthritic knee joint. METHODS: This study involves a prospective, randomized, placebo-controlled, and single-blind trial based on the SPIRIT guidelines, and aims to recruit 96 patients initially diagnosed with knee osteoarthritis, following American College of Rheumatology criteria. Patients will be randomized in a 1:1:1 ratio to receive Intraarticular HADMSCs injection with LIPUS, Intraarticular HADMSCs injection with shame LIPUS, or Normal saline with LIPUS. The primary outcome is Western Ontario and McMaster Universities Index of OA (WOMAC) score, while the secondary outcomes will be other knee structural changes, and lower limb muscle strength such as the knee cartilage thickness measured by MRI. Blinded assessments will be performed at baseline (1 month prior to treatment), 1 month, 3 months, and 6 months following the interventions. DISCUSSION: This trial will be the first clinical study to comprehensively investigate the safety and efficacy of LIPUS on stem cell therapy in OA patients. The results may provide evidence of the effectiveness of LIPUS in improving stem cell therapy and deliver valuable information for the design of subsequent trials. TRIAL REGISTRATION: This study had been prospectively registered with the Chinese Clinical Trials Registry. registration number: ChiCTR1900025907 at September 14, 2019.

Fucoidan exerts antidepressant-like effects in mice via regulating the stability of surface AMPARs.

The inflammatory hypothesis is one of the most important mechanisms of depression. Fucoidan is a bioactive sulfated polysaccharide abundant in brown seaweeds with anti-inflammatory activity. However, the antidepressant effects of fucoidan on chronic stress-induced depressive-like behaviors have not been well elucidated. Here, we used two different depressive-like mouse models, lipopolysaccharide (LPS) and chronic restraint stress (CRS) models, to explore the detailed molecular mechanism underlying its antidepressant-like effects in C57BL/6J mice by combining multiple behavioral, molecular and immunofluorescence experiments. Adenovirus-mediated overexpression of caspase-1 and pharmacological inhibitors were also used to clarify the antidepressant mechanisms of fucoidan. We found that acute administration of fucoidan did not produce antidepressant effects in the tail suspension test (TST) and forced swim test (FST). Interestingly, chronic fucoidan administration not only dose-dependently reduced stress-induced depressive-like behaviors in the TST, FST, sucrose preference test (SPT), and novelty-suppressed feeding test (NSFT), but also alleviated the downregulation of brain-derived neurotrophic factor (BDNF)-dependent synaptic plasticity via inhibiting caspase-1-mediated inflammation in the hippocampus of mice. Moreover, fucoidan significantly ameliorated behavioral and synaptic plasticity abnormalities in the overexpression of caspase-1 in the hippocampus of mice. Furthermore, blocking BDNF abolished the antidepressant-like effects of fucoidan in mice. Therefore, our findings clearly indicate that fucoidan provides a potential supplementary noninvasive treatment for depression by inhibition of hippocampal inflammation.

Increased Homer1-mGluR5 mediates chronic stress-induced depressive-like behaviors and glutamatergic dysregulation via activation of PERK-eIF2α.

Glutamatergic dysregulation has served as one common pathophysiology of major depressive disorder (MDD) and a promising target for treatment intervention. Previous studies implicate neurotransmission via metabotropic glutamate receptors (mGluRs) and Homer1 in stress-induced anhedonia, but the mechanisms have not been well elucidated. In the present study, we used two different animal models of depression, chronic social defeat stress (CSDS) and chronic restraint stress (CRS), to investigate the expression of Homer1 isoforms and functional interaction with mGluRs. We found that chronic stress selectively upregulated the expression of Homer1b/c in the hippocampus, whereas the level of Homer1a was unchanged. Additionally, there was a significant negative correlation between the levels of Homer1-mGluR5 signaling and depressive-like behaviors. Both application of paired-pulse low-frequency stimulation (PP-LFS) and the selective group 1 mGluRs agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) significantly enhanced mGluR-dependent long-term depression (LTD) at CA3-CA1 pyramidal cell synapses in slices from susceptible mice, whereas there was no change in NMDAR-dependent LTD induced by LFS. Furthermore, these effects were associated with the internalization of surface AMPARs in hippocampal pyramidal neurons, including reduced the expression of AMPARs and amplitude of AMPARs-mediated mEPSC. Finally, we found that chronic stress activated the KR-like ER kinase-eukaryotic initiation factor 2α (PERK-eIF2α) signaling pathway, subsequently phosphorylated cAMP response element binding protein (CREB) at the S129 and reduced the BDNF level, eventually leading to the impairment of synaptic transmission and depressive-like behaviors. Therefore, our study suggests that PERK-eIF2α acts as a critical target downstream of Homer1-mGluR5 complex to mediate chronic stress-induced depressive-like behaviors, and highlights them as a potential target for the treatment of mood disorder.