Defining recovery neurobiology of injured spinal cord by synthetic matrix-assisted hMSC implantation.

Mesenchymal stromal stem cells (MSCs) isolated from adult tissues offer tangible potential for regenerative medicine, given their feasibility for autologous transplantation. MSC research shows encouraging results in experimental stroke, amyotrophic lateral sclerosis, and neurotrauma models. However, further translational progress has been hampered by poor MSC graft survival, jeopardizing cellular and molecular bases for neural repair in vivo. We have devised an adult human bone marrow MSC (hMSC) delivery formula by investigating molecular events involving hMSCs incorporated in a uniquely designed poly(lactic-co-glycolic) acid scaffold, a clinically safe polymer, following inflammatory exposures in a dorsal root ganglion organotypic coculture system. Also, in rat T9-T10 hemisection spinal cord injury (SCI), we demonstrated that the tailored scaffolding maintained hMSC stemness, engraftment, and led to robust motosensory improvement, neuropathic pain and tissue damage mitigation, and myelin preservation. The scaffolded nontransdifferentiated hMSCs exerted multimodal effects of neurotrophism, angiogenesis, neurogenesis, antiautoimmunity, and antiinflammation. Hindlimb locomotion was restored by reestablished integrity of submidbrain circuits of serotonergic reticulospinal innervation at lumbar levels, the propriospinal projection network, neuromuscular junction, and central pattern generator, providing a platform for investigating molecular events underlying the repair impact of nondifferentiated hMSCs. Our approach enabled investigation of recovery neurobiology components for injured adult mammalian spinal cord that are different from those involved in normal neural function. The uncovered neural circuits and their molecular and cellular targets offer a biological underpinning for development of clinical rehabilitation therapies to treat disabilities and complications of SCI.

Alleviation of chronic pain following rat spinal cord compression injury with multimodal actions of huperzine A.

Diverse mechanisms including activation of NMDA receptors, microglial activation, reactive astrogliosis, loss of descending inhibition, and spasticity are responsible for ∼40% of cases of intractable neuropathic pain after spinal cord injury (SCI). Because conventional treatments blocking individual mechanisms elicit only short-term effectiveness, a multimodal approach with simultaneous actions against major pain-related pathways may have value for clinical management of chronic pain. We hypothesize that [-]-huperzine A (HUP-A), an alkaloid isolated from the club moss Huperzia serrata, that is a potent reversible inhibitor of acetylcholinesterase and NMDA receptors, could mitigate pain without invoking drug tolerance or dependence by stimulating cholinergic interneurons to impede pain signaling, inhibiting inflammation via microglial cholinergic activation, and blocking NMDA-mediated central hypersensitization. We tested our hypothesis by administering HUP-A i.p. or intrathecally to female Sprague-Dawley rats (200-235 g body weight) after moderate static compression (35 g for 5 min) of T10 spinal cord. Compared with controls, HUP-A treatment demonstrates significant analgesic effects in both regimens. SCI rats manifested no drug tolerance following repeated bolus i.p. or chronic intrathecal HUP-A dosing. The pain-ameliorating effect of HUP-A is cholinergic dependent. Relative to vehicle treatment, HUP-A administration also reduced neural inflammation, retained higher numbers of calcium-impermeable GluR2-containing AMPA receptors, and prevented Homer1a up-regulation in dorsal horn sensory neurons. Therefore, HUP-A may provide safe and effective management for chronic postneurotrauma pain by reestablishing homeostasis of sensory circuits.

Physical impacts of PLGA scaffolding on hMSCs: Recovery neurobiology insight for implant design to treat spinal cord injury.

Our earlier work generated a powerful platform technology of polymeric scaffolding of stem cells to investigate and treat the injured or diseased central nervous system. However, the reciprocal sequelae between biophysical properties of the polymer and responses of the stem cell have not been examined in situ in lesioned spinal cords. We postulated that implantable synthetic scaffolds, acting through physical features, might affect donor cell behavior and host tissue remodeling. To test this hypothesis, poly(d,l-lactic-co-glycolic acid) (PLGA) in either low/soft or high/hard rigidity was fabricated for carrying adult human bone marrow mesenchymal stromal stem cells (hMSCs). The construct was transplanted into the epicenter of a rat model of acute T9-10 segmental hemisection to evaluate the effect of PLGA rigidity on the therapeutic potential and fate of hMSCs for neural repair. Compared to controls, only treatment with soft PLGA-scaffolded hMSCs significantly improved sensorimotor function via activation of recovery neurobiology mechanisms. The main benefits included inhibiting neuroinflammation and enhancing tissue protection. Also detected in the treated lesion region were expressions of neurotrophic and anti-inflammatory factors together with proliferation of endogenous neural stem cells, impacts likely derived from hMSCs' functional multipotency maintained by soft PLGA-scaffolding. Conversely, hard rigidity PLGA activated mechanotransduction and mesoderm lineage differentiation of hMSCs that ectopically produced bone, cartilage and muscle markers in neural parenchyma. The findings collectively suggested that the physical texture of polymeric scaffolds should be tailored for sustaining the stemness of hMSCs to constructively interact with the spinal cord for functional restoration.

Establishing an Organotypic System for Investigating Multimodal Neural Repair Effects of Human Mesenchymal Stromal Stem Cells.

Human mesenchymal stromal stem cells (hMSCs) hold regenerative medicine potential due to their availability, in vitro expansion readiness, and autologous feasibility. For neural repair, hMSCs show translational value in research on stroke, spinal cord injury (SCI), and traumatic brain injury. It is pivotal to establish multimodal in vitro systems to investigate molecular mechanisms underlying neural actions of hMSCs. Here, we describe a platform protocol on how to set up organotypic co-cultures of hMSCs (alone or polymer-scaffolded) with explanted adult rat dorsal root ganglia (DRGs) to determine neural injury and recovery events for designing implants to counteract neurotrauma sequelae. We emphasize in vitro hMSC propagation, polymer scaffolding, hMSC stemness maintenance, hMSC-DRG interaction profiling, and analytical formulas of neuroinflammation, trophic factor expression, DRG neurite outgrowth and tropic tracking, and in vivo verification of tailored implants in rodent models of SCI. © 2018 by John Wiley & Sons, Inc.

Downregulation of extracellular matrix-related gene clusters during osteogenic differentiation of human bone marrow- and adipose tissue-derived stromal cells.

Bone marrow- and adipose tissue-derived stromal cells (BMSCs and ASCs, respectively) exhibit a similar capacity for osteogenic differentiation in vitro, but it is unclear whether they share a common differentiation process, because they originate from different tissues. The aim of this study was to explore BMSC and ASC osteogenic differentiation by focusing on the expression of extracellular matrix-related genes (ECMGs), which play a crucial role in osteogenesis and bone tissue regeneration in vivo. We characterized the gene expression profiles of BMSCs and ASCs using a custom complementary deoxyribonucleic acid microarray containing 55 ECMGs. Undifferentiated BMSCs and ASCs actively expressed a wide range of ECMGs. Once BMSCs and ASCs were placed in an osteogenic differentiation medium, 24 and 17 ECMGs, respectively, underwent considerable downregulation over the course of the culture period. The remaining genes were maintained at a similar expression level to corresponding uninduced cell cultures. Although the suppression phenomenon was consistent irrespective of stromal cell origin, collagen (COL)2A1, COL6A1, COL9A1, parathyroid hormone receptor, integrin (INT)-beta3, and TenascinX genes were only downregulated in osteogenic BMSCs, whereas COL1A2, COL3A1, COL4A1, COL5A2, COL15A1, osteopontin, osteonectin, and INT-beta1 genes were only downregulated in osteogenic ASCs. During this time period, cell viability was sustained, suggesting that the observed downregulation did not occur by selection and elimination of unfit cells from the whole cell population. These data suggest that osteogenically differentiating BMSCs and ASCs transition away from a diverse gene expression pattern, reflecting their multipotency toward a configuration specifically meeting the requirements of the target lineage. This change may serve to normalize gene expression in mixed populations of stem cells derived from different tissues.

Nontoxic genetic engineering of mesenchymal stem cells using serum-compatible pullulan-spermine/DNA anioplexes.

Genetic modification of stem cells could be applied to initiate/enhance their secretion of therapeutic molecules, alter their biological properties, or label them for in vivo tracking. We recently developed a negatively charged gene carrier ("anioplex") based on pullulan-spermine, a conjugate prepared from a natural polysaccharide and polyamine. In rat mesenchymal stem cells (MSCs), anioplex-derived reporter gene activity was comparable to or exceeded that obtained using a commercial cationic lipid reagent. Transfection in the growth medium with 15% serum and antibiotics was approximately sevenfold more effective than in serum-free conditions. Cytotoxicity was essentially indiscernible after 24 h of anioplex transfection with 20 µg/mL DNA, in contrast to cationic lipid transfection that resulted in 40%-60% death of target MSCs. Anioplex-derived reporter gene activity persisted throughout the entire 3-week study, with post-transfection MSCs appearing to maintain osteogenic, adipogenic, and chondrogenic multipotency. In particular, chondrogenic pellet formation of differentiating human MSCs was significantly inhibited after lipofection but not after aniofection, which further indicates the biological inertness of pullulan-spermine/DNA anioplexes. Collectively, these data introduce a straightforward technology for genetic engineering of adult stem/progenitor cells under physiological niche-like conditions. Moreover, reporter gene activity was observed in rat spinal cords after minimally invasive intrathecal implantation, suggesting effective engraftment of donor MSCs. It is therefore plausible that anioplex-transfected MSCs or other stem/progenitor cells with autologous potential could be applied to disorders such as neurotrauma or neuropathic pain that involve the spinal cord and brain.

Neuronal gene delivery by negatively charged pullulan-spermine/DNA anioplexes.

Nonviral gene transfer to neurons remains unreliable due to a lack of effective and nontoxic vectors. Here, we achieved effective neuronal gene delivery through salt-free complexation of plasmid DNA and pullulan-spermine, a conjugate prepared from a naturally derived polysaccharide and polyamine. Specifically, at low spermine nitrogen:DNA phosphate (N:P) ratios, complexes formed with zeta-potential and diameter of approximately-40mV and 350nm, respectively. Higher N:P ratios increased the zeta-potential to approximately +10mV. All complexes were stable for at least 1 week and protected DNA from degradation. In vitro transfection of rat sensory neurons occurred at all N:P ratios, but uniquely, efficiency was highest for anionic complexes (anioplexes). Subsequent analyses revealed the inhibition of reporter gene expression by asialofetuin (1mg/ml) and methyl-beta-cyclodextrin (5mm), indicating utilization of glycoprotein-specific interactions and lipid rafts for uptake and intracellular trafficking. In marked contrast to a commercial cationic lipid reagent, anioplexes did not exhibit measurable cytotoxicity at up to 20microg/ml DNA. Additionally, transfection efficiency was maintained in the presence of serum and antibiotics. Based on these favorable properties, we successfully established two transfection methods for cultured adult sensory neurons and tissue explants. Collectively, these data suggest that negatively charged pullulan-spermine/DNA anioplexes could represent an effective gene delivery technology, particularly for neurons.

Enhanced bone regeneration via multimodal actions of synthetic peptide SVVYGLR on osteoprogenitors and osteoclasts.

Recently, the binding sequence Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR) was found adjacent to the RGD sequence in osteopontin, suggesting involvement in osteo-immune cross-talk. The aim of this study was to investigate bioactive functions of a synthetic SVVYGLR peptide in osteoprogenitor cells and osteoclasts, and to examine potential applications in bone regeneration. The SVVYGLR peptide significantly enhanced the adhesion and proliferation of several human mesenchymal cells including bone marrow-derived mesenchymal stem cells, and alphavbeta3 integrin was involved in cell attachment to the peptide. Additionally, the peptide reduced the number of TRAP-positive multinucleated cells during osteoclastogenesis of RAW264.7 cells and normal murine pre-osteoclasts, and also suppressed NFAT activity and expression of osteoclastogenesis-related mRNAs. When standardized bone defects in rat calvariae were filled with a collagen sponge containing the peptide or PBS (control), the number of TRAP-positive osteoclasts in the grafted sites after 3 weeks was significantly lower in the peptide group. By the 5th week, significantly enhanced resorption of the grafted collagen sponge and new bone formation was observed within and surrounding the sponge in the peptide group. These data suggest that SVVYGLR is an effective bioactive peptide for bone tissue regeneration that promotes attachment and proliferation of osteogenic cells while also suppressing osteoclastogenesis.