Chen Huan Lin

Intracerebral implantation of autologous peripheral blood stem cells in stroke patients: A randomized phase II study

In our previous study, intracerebral implantation of peripheral blood stem cells (PBSCs) improved functional outcome in rats with chronic cerebral infarction. Based on this finding, a randomized, single blind controlled study was conducted in 30 patients [PBSC group (n = 15) and control group (n = 15)] with middle cerebral artery infarction confirmed on a T2-weighted MRI 6 months to 5 years after a stroke. Only subjects with neurological deficits of intermediate severity based on the National Institute of Health Stroke Scale (NIHSS; range: 9–20) that had been stable for at least 3 months were enrolled. Those in the PBSC group received subcutaneous G-CSF injections (15 μg/kg/day) for 5 consecutive days, and then stereotaxic implantation of 3–8 × 106 CD34+ immunosorted PBSCs. All 30 patients completed the 12-month follow-up. No serious adverse events were noted during study period. Improvements in stroke scales (NIHSS, ESS, and EMS) and functional outcomes (mRS) from baseline to the end of the 12-month follow-up period were significantly greater in the PBSC than the control group. The fiber numbers asymmetry (FNA) scores based on diffusion tensor image (DTI) tractography were reduced in every PBSC-treated subject, but not in the control group. Reduction in the FNA scores correlated well with the improvement in NIHSS. Furthermore, a positive motor-evoked potential (MEP) response by transcranial magnetic stimulation (TMS) appeared in 9 of the 15 subjects in the PBSC group. This phase II study demonstrated that implantation of autologous CD34+ PBSC was safe, feasible, and effective in improving functional outcome.

Multitheragnostic Multi-GNRs Crystal-Seeded Magnetic Nanoseaurchin for Enhanced In Vivo Mesenchymal-Stem-Cell Homing, Multimodal Imaging, and Stroke Therapy.

A multifunctional nanoseaurchin probe in which mesoporous silica nanobeads with iron oxide nanoparticles embedded and multi-gold nanorods crystal-seeded are fabricated and labeled with umbilical cord mesenchymal stem cells through endocytosis. This nanoplatform enables efficient magnetic remote-controlled guiding for stem cell homing, and provides dual modalities of photoacoustic imaging and magnetic resonance imaging for in situ tracking and long-term monitoring to achieve therapeutic efficacy.

Neural stem cells and stroke

Acute ischemic stroke causes a disturbance of neuronal circuitry and disruption of the blood–brain barrier that can lead to functional disabilities. At present, thrombolytic therapy inducing recanalization of the occluded vessels in the cerebral infarcted area is a commonly used therapeutic strategy. However, only a minority of patients have timely access to this kind of therapy. Recently, neural stem cells (NSCs) as therapy for stroke have been developed in preclinical studies. NSCs are harbored in the subventricular zone (SVZ) as well as the subgranular zone of the brain. The microenvironment in the SVZ, including intercellular interactions, extracellular matrix proteins, and soluble factors, can promote NSC proliferation, self-renewal, and multipotency. Endogenous neurogenesis responds to insults of ischemic stroke supporting the existence of remarkable plasticity in the mammalian brain. Homing and integration of NSCs to the sites of damaged brain tissue are complex morphological and physiological processes. This review provides an update on current preclinical cell therapies for stroke, focusing on neurogenesis in the SVZ and dentate gyrus and on recruitment cues that promote NSC homing and integration to the site of the damaged brain.

Role of HIF-1α-activated Epac1 on HSC-mediated neuroplasticity in stroke model

Exchange protein activated by cAMP-1 (Epac1) plays an important role in cell proliferation, cell survival and neuronal signaling, and activation of Epac1 in endothelial progenitor cells increases their homing to ischemic muscles and promotes neovascularization in a model of hind limb ischemia. Moreover, upregulation of Epac1 occurs during organ development and in diseases such as myocardial hypertrophy, diabetes, and Alzheimers disease. We report here that hypoxia upregulated Epac1 through HIF-1α induction in the CD34-immunosorted human umbilical cord blood hematopoietic stem cells (hUCB(34)). Importantly, implantation of hUCB(34) subjected to hypoxia-preconditioning (HP-hUCB(34)) improved stroke outcome, more than did implantation of untreated hUCB(34), in rodents subjected to cerebral ischemia, and this required Epac1-to-matrix metalloprotease (MMP) signaling. This improved therapeutic efficacy correlated with better engraftment and differentiation of these cells in the ischemic host brain. In addition, more than did implantation of untreated HP-hUCB(34), implantation of HP-hUCB(34) improved cerebral blood flow into the ischemic brain via induction of angiogenesis, facilitated proliferation/recruitment of endogenous neural progenitor cells in the ischemic brain, and promoted neurite outgrowth following cerebral ischemia. Consistent with our proposed role of Epac1-to-MMP signaling in hypoxia-preconditioning, the above mentioned effects of implanting HP-hUCB(34) could be abolished by pharmacological inhibition and genetic disruption/deletion of Epac1 or MMPs. We have discovered a HIF-1α-to-Epac1-to-MMP signaling pathway that is required for the improved therapeutic efficacy resulting from hypoxia preconditioning of hUCB(34) in vitro prior to their implantation into the host brain in vivo.

Role of stress‐inducible protein‐1 in recruitment of bone marrow derived cells into the ischemic brains

Stress‐inducible protein‐1 (STI‐1) is the proposed ligand for the cellular prion protein (PrPC), which is thought to facilitate recovery following stroke. Whether STI‐1 expression is affected by stroke and how its signalling facilitates recovery remain elusive. Brain slices from patients that died of ischemic stroke were collected for STI‐1 immunohistochemistry. These findings were compared to results from cell cultures, mice with or without the PrPC knockout, and rats. Based on these findings, molecular and pharmacological interventions were administered to investigate the underlying mechanisms and to test the possibility for therapy in experimental stroke models. STI‐1 was upregulated in the ischemic brains from humans and rodents. The increase in STI‐1 expression in vivo was not cell‐type specific, as it was found in neurons, glia and endothelial cells. Likewise, this increase in STI‐1 expression can be mimicked by sublethal hypoxia in primary cortical cultures (PCCs) in vitro, and appear to have resulted from the direct binding of the hypoxia inducible factor‐1α (HIF‐1α) to the STI‐1 promoter. Importantly, this STI‐1 signalling promoted bone marrow derived cells (BMDCs) proliferation and migration in vitro and recruitment to the ischemic brain in vivo, and augmenting its signalling facilitated neurological recovery in part by recruiting BMDCs to the ischemic brain. Our results thus identified a novel mechanism by which ischemic insults can trigger a self‐protective mechanism to facilitate recovery.

PACAP38/PAC1 signaling induces bone marrow-derived cells homing to ischemic brain

Understanding stem cell homing, which is governed by environmental signals from the surrounding niche, is important for developing effective stem cell‐based repair strategies. The molecular mechanism by which the brain under ischemic stress recruits bone marrow‐derived cells (BMDCs) to the vascular niche remains poorly characterized. Here we report that hypoxia‐inducible factor‐1α (HIF‐1α) activation upregulates pituitary adenylate cyclase‐activating peptide 38 (PACAP38), which in turn activates PACAP type 1 receptor (PAC1) under hypoxia in vitro and cerebral ischemia in vivo. BMDCs homing to endothelial cells in the ischemic brain are mediated by HIF‐1α activation of the PACAP38‐PAC1 signaling cascade followed by upregulation of cellular prion protein and α6‐integrin to enhance the ability of BMDCs to bind laminin in the vascular niche. Exogenous PACAP38 confers a similar effect in facilitating BMDCs homing into the ischemic brain, resulting in reduction of ischemic brain injury. These findings suggest a novel HIF‐1α‐activated PACAP38‐PAC1 signaling process in initiating BMDCs homing into the ischemic brain for reducing brain injury and enhancing functional recovery after ischemic stroke. Stem Cells 2015;33:1153–1172

A crucial role of CXCL14 for promoting regulatory T cells activation in stroke

Inflammatory processes have a detrimental role in the pathophysiology of ischemic stroke. However, little is known about the endogenous anti-inflammatory mechanisms in ischemic brain. Here, we identify CXCL14 as a critical mediator of these mechanisms. CXCL14 levels were upregulated in the ischemic brains of humans and rodents. Moreover, hypoxia inducible factor-1α (HIF-1α) drives hypoxia- or cerebral ischemia (CI)-dependent CXCL14 expression via directly binding to the CXCL14 promoter. Depletion of CXCL14 inhibited the accumulation of immature dendritic cells (iDC) or regulatory T cells (Treg) and increased the infarct volume, whereas the supplementation of CXCL14 had the opposite effects. CXCL14 promoted the adhesion, migration, and homing of circulating CD11c+ iDC to the ischemic tissue via the upregulation of the cellular prion protein (PrPC), PECAM-1, and MMPs. The accumulation of Treg in ischemic areas of the brain was mediated through a cooperative effect of CXCL14 and iDC-secreted IL-2-induced Treg differentiation. Interestingly, CXCL14 largely promoted IL-2-induced Treg differentiation. These findings indicate that CXCL14 is a critical immunomodulator involved in the stroke-induced inflammatory reaction. Passive CXCL14 supplementation provides a tractable path for clinical translation in the improvement of stroke-induced neuroinflammation.

Role of IGF1R+ MSCs in modulating neuroplasticity via CXCR4 cross-interaction

To guide the use of human mesenchymal stem cells (MSCs) toward clinical applications, identifying pluripotent-like-markers for selecting MSCs that retain potent self-renewal-ability should be addressed. Here, an insulin-like growth factor 1 receptor (IGF1R)–expressing sub-population in human dental pulp MSCs (hDSCs), displayed multipotent properties. IGF1R expression could be maintained in hDSCs when they were cultured in 2% human cord blood serum (hUCS) in contrast to that in 10% fetal calf serum (FCS). Cytokine array showed that hUCS contained higher amount of several growth factors compared to FCS, including IGF-1 and platelet-derived growth factor (PDGF-BB). These cytokines modulates the signaling events in the hDSCs and potentially enhances engraftment upon transplantation. Specifically, a bidirectional cross-talk between IGF1R/IGF1 and CXCR4/SDF-1α signaling pathways in hDSCs, as revealed by interaction of the two receptors and synergistic activation of both signaling pathways. In rat stroke model, animals receiving IGF1R+ hDSCs transplantation, interaction between IGF1R and CXCR4 was demonstrated to promote neuroplasticity, therefore improving neurological function through increasing glucose metabolic activity, enhancing angiogenesis and anti-inflammatiory effects. Therefore, PDGF in hUCS-culture system contributed to the maintenance of the expression of IGF1R in hDSCs. Furthermore, implantation of IGF1R+ hDSCs exerted enhanced neuroplasticity via integrating inputs from both CXCR4 and IGF1R signaling pathways.

IGF1R+ Dental Pulp Stem Cells Enhanced Neuroplasticity in Hypoxia-Ischemia Model

Until now, the surface markers of multipotent mesenchymal stem cells (MSCs) had not been fully identified. Here, we found that the IGF1 receptor (IGF1R), regarded as a pluripotent marker of embryonic stem cells (ESCs), was also expressed in human dental pulp derived-mesenchymal stem cells (hDSCs), which displayed a potential for both self-renewal and multipotency. hDSC-secreted IGF1 interacted with IGF1R through an autocrine signaling pathway to maintain this self-renewal and proliferation potential. Stereotaxic implantation of immunosorted IGF1R+ hDSCs in rats with neonatal hypoxia-ischemia (NHI) promoted neuroplasticity, improving the neurological outcome by increasing expression of the anti-apoptotic protein Bcl-2, which enhanced both neurogenesis and angiogenesis. In addition, treatment with IGF1R+ hDSCs significantly modulated neurite regeneration and anti-inflammation in vivo in NHI rats and in vitro in primary cortical cultures under oxygen/glucose deprivation. Autocrine regulatory expression of IGF1R contributed to maintaining the self-renewal capacity of hDSCs. Furthermore, implantation of IGF1R+ hDSCs increased neuroplasticity with neurite regeneration and immunomodulation in and the NHI rat model.

Increase of Meningitis Risk in Stroke Patients in Taiwan

Background and purpose The blood–brain barrier (BBB) not only provides a physical obstruction but also recruits and activates neutrophils in cases of infection. Hemorrhagic or ischemic stroke reportedly induces the disruption of the BBB. However, few studies have reported a correlation between the incidence of meningitis in patients with a history of stroke. This study tested the hypothesis that patients with a history of stroke may be more vulnerable to meningitis. Methods Stroke and age-matched comparison (n = 29,436 and 87,951, respectively) cohorts were recruited from the Taiwan National Health Insurance database (2000–2011). Correlations between the two cohorts were evaluated by Cox proportional hazard regression model, Kaplan–Meier curve, and log-rank tests. Results The incidence of meningitis was higher in the stroke cohort compared to that in the comparison cohort [hazard ratio (HR), 2.89; 95% confidence interval (CI), 2.23–3.74, p < 0.001]. After adjusting for age, sex, and comorbidities, the estimated HR in the stroke cohort was 2.55-fold higher than that in the comparison cohort (CI, 1.94–3.37; p < 0.001). Notably, patients who had experienced hemorrhagic stroke had a higher incidence rate of meningitis than those with a history of ischemic stroke, except for patients older than 75 years (incidence rates in hemorrhagic/ischemic stroke patients, 3.14/1.48 in patients younger than 45 years, 1.52/0.41 in 45- to 64-year group, 1.15/0.90 in 65- to 74-year group, 0.74/0.93 in patients older than 75 years). Moreover, stroke patients who had undergone head surgery had the highest meningitis risk (adjusted HR, 8.66; 95% CI, 5.55–13.5; p < 0.001) followed by stroke patients who had not undergone head surgery (adjusted HR, 2.11; 95% CI, 1.57–2.82; p < 0.001). Conclusion Our results indicated that stroke patients have higher risks of meningitis. Compromised BBB integrity in stroke patients may lead to increased vulnerability to infectious pathogens. In summary, our study provided new evidence of the clinical relationship between stroke and meningitis, and our findings suggest the need for precautions to prevent meningitis in stroke patients.