Investigation of mesenchymal stem cells and the development of experimental strategies for rescuing glaucomatous eyes using a stem cell-based therapy

Glaucoma, the second leading cause of blindness in the world, is a hereditary ocular disease and about 60 million people suffer from it. However, currently there are no remedies to cure glaucoma. As a potential solution to rescue degenerating retinas, stem cells have been investigated extensively and the effects of morphological neuroprotection or functional recovery have been reported. In the present studies, I aimed to investigate two main topics of research. From the first topic, we demonstrated MSCs from Brown Norway rats are a self-renewing, multi-potent population. More importantly, we provide the first evidence that bone marrow MSCs are capable of promoting neurite outgrowth from adult hippocampal progenitor cells (AHPCs). For the second topic of research, mouse MSCs were engineered to over-express brain-derived neurotrophic factor (BDNF) and/or glial cell-derived neurotrophic factor (GDNF) together with green fluorescent protein (GFP), as a strategy for stem cell-based therapy. Our results show MSCs, infected with lentiviral vectors encoding BDNF or GDNF, noticeably increased the release of bioactive neurotrophic factors in vitro. For our in vivo study of stem cell-therapy, two different mouse models of experimental glaucoma were studied. MSCs, following intraocular transplantation, survived up to 4 months post-transplant (PT) in the host retina of adult DBA/2J mice. Interestingly, our results indicated MSCs expressing GDNF played a potential role in alleviating glial activation on host retinas. Importantly, our data show GDNF/BDNF-MSCs contributed to preservation of the inner retina of the recipient eye, compared to the control fellow eye. An additional transgenic mouse model of glaucoma, Myocilin (MYOC)-mutant mice, was investigated. Our preliminary study showed transplanted MSCs survived up to 11.5 months-PT in the adult host retina. No significant changes in the number of retinal ganglion cells between experimental and control eyes were found at 11.5 months-PT. These studies are the first to characterize bone marrow-derived MSCs from Brown Norway rats and to study potential neuroprotective effects of transplanted MSCs on the host retinas of DBA/2J and MYOC-mice. This study provides an experimental strategy towards clinical applications in terms of using MSCs as a potential autograft and long-term neuroprotection via neurotrophic factor delivery for retinodegenerative diseases

Bone marrow-derived mesenchymal stem cells (MSCs) stimulate neurite outgrowth from differentiating adult hippocampal progenitor cells

Background: Bone marrow-derived mesenchymal stem cells (MSCs) have emerged as beneficial cellular vehicles for nervous system rescue and repair. A better understanding how MSCs are involved in mediating neural repair will facilitate development of novel therapeutic strategies. Methods: In the present study bone marrow-derived MSCs were isolated and characterized from Brown Norway rats. The capacity of the MSCs to influence the differentiation of adult hippocampal progenitor cells (AHPCs) was investigated using contact and non-contact co-culture configurations. Results: These MSCs showed a stable and consistent growth rate, retained short population doubling time (PDT) and showed high capacity of cell proliferation. Co-culturing of AHPCs with MSCs did not appear to significantly affect the proliferation of the AHPCs or impact the proportion of neuronal or glial differentiation of the AHPCs. However, both contact co-culture (CCC) and non-contact co-culture (NCCC) significantly promoted neurite outgrowth from neuronal AHPCs. Conclusions: The ability of MSCs to promote the morphological differentiation of AHPCs may serve as an added benefit when developing cell-based strategies for nervous system rescue and repair

Multipotent adult hippocampal progenitor cells maintained as neurospheres favor differentiation toward glial lineages

Adult hippocampal progenitor cells (AHPCs) are generally maintained as a dispersed monolayer population of multipotent neural progenitors. To better understand cell-cell interactions among neural progenitors and their influences on cellular characteristics, we generated free-floating cellular aggregates, or neurospheres, from the adherent monolayer population of AHPCs. Results from in vitro analyses demonstrated that both populations of AHPCs were highly proliferative under maintenance conditions, but AHPCs formed in neurospheres favored differentiation along a glial lineage and displayed greater migrational activity, than the traditionally cultured AHPCs. To study the plasticity of AHPCs from both populations in vivo, we transplanted GFP-expressing AHPCs via intraocular injection into the developing rat eyes. Both AHPC populations were capable of surviving and integrating into the developing host central nervous system, but considerably more GFP-positive cells were observed in the retinas transplanted with neurosphere AHPCs, compared to adherent AHPCs. These results suggest that the culture configuration during maintenance for neural progenitor cells (NPCs) influences cell fate and motility in vitro as well as in vivo. Our findings have implication for understanding different cellular characteristics of NPCs according to distinct intercellular architectures and for developing cell-based therapeutic strategies using lineage-committed NPCs

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system. Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in longterm in vivo transplant studies for nervous system rescue and repair

A Protective Role for Heme Oxygenase-1 in INS-1 Cells and Rat Islets that are Exposed to High Glucose Conditions

Heme oxygenase-1 (HO-1) has been described as an inducible protein that is capable of cytoprotection via radical scavenging and the prevention of apoptosis. Chronic exposure to hyperglycemia can lead to cellular dysfunction that may become irreversible over time, and this process has been termed glucose toxicity. Yet little is known about the relation between glucose toxicity and HO-1 in the islets. The purposes of the present study were to determine whether prolonged exposure of pancreatic islets to a supraphysiologic glucose concentration disrupts the intracellular balance between reactive oxygen species (ROS) and HO-1, and so this causes defective insulin secretion; we also wanted to evaluate a protective role for HO-1 in pancreatic islets against high glucose levels. The intracellular peroxide levels of the pancreatic islets (INS-1 cell, rat islet) were increased in the high glucose media (30 mM glucose or 50 mM ribose). The HO-1 expression was induced in the INS-1 cells by the high glucose levels. Both the HO-1 expression and glucose stimulated insulin secretion (GSIS) was decreased simultaneously in the islets by treatment of the HO-1 antisense. The HO-1 was upregulated in the INS-1 cells by hemin, an inducer of HO-1. And, HO-1 upregulation induced by hemin reversed the GSIS in the islets at a high glucose condition. These results suggest HO-1 seems to mediate the protective response of pancreatic islets against the oxidative stress that is due to high glucose conditions

Molecular diagnosis of hereditary spherocytosis by multi-gene target sequencing in Korea: matching with osmotic fragility test and presence of spherocyte

Background Current diagnostic tests for hereditary spherocytosis (HS) focus on the detection of hemolysis or indirectly assessing defects of membrane protein, whereas direct methods to detect protein defects are complicated and difficult to implement. In the present study, we investigated the patterns of genetic variation associated with HS among patients clinically diagnosed with HS. Methods Multi-gene targeted sequencing of 43 genes (17 RBC membrane protein-encoding genes, 20 RBC enzyme-encoding genes, and six additional genes for the differential diagnosis) was performed using the Illumina HiSeq platform. Results Among 59 patients with HS, 50 (84.7%) had one or more significant variants in a RBC membrane protein-encoding genes. A total of 54 significant variants including 46 novel mutations were detected in six RBC membrane protein-encoding genes, with the highest number of variants found in SPTB (n = 28), and followed by ANK1 (n = 19), SLC4A1 (n = 3), SPTA1 (n = 2), EPB41 (n = 1), and EPB42 (n = 1). Concurrent mutations of genes encoding RBC enzymes (ALDOB, GAPDH, and GSR) were detected in three patients. UGT1A1 mutations were present in 24 patients (40.7%). Positive rate of osmotic fragility test was 86.8% among patients harboring HS-related gene mutations. Conclusions This constitutes the first large-scaled genetic study of Korean patients with HS. We demonstrated that multi-gene target sequencing is sensitive and feasible that can be used as a powerful tool for diagnosing HS. Considering the discrepancies of clinical and molecular diagnoses of HS, our findings suggest that molecular genetic analysis is required for accurate diagnosis of HS.Support was provided by: the National Research Foundation of Korea (NRF) grant funded by the Korea government(MSIT) (NRF-2017R1A2A1A17069780) http://www.nrf.re.kr/

The Therapeutic Effect of STAT3 Signaling-Suppressed MSC on Pain and Articular Cartilage Damage in a Rat Model of Monosodium Iodoacetate-Induced Osteoarthritis

Osteoarthritis (OA) is a degenerative disease that induces pain, cartilage deformation, and joint inflammation. Mesenchymal stem cells (MSCs) are potential therapeutic agents for treatment of OA. However, MSC therapy can cause excessive inflammation. Signal transducer and activator of transcription 3 (STAT3) modulates secretion of many proinflammatory cytokines. Experimental OA was induced by intra-articular (IA) injection of monosodium iodoacetate (MIA) to the right knee of rats. MSCs from OA patients (OA-MSCs) were treated with STA21, a small molecule that blocks STAT3 signaling, by IA or intravenous (IV) injection after MIA injection. Pain severity was quantified by assessment of secondary tactile allodynia using the von Frey assessment test. Cartilage degradation was measured by microcomputed tomography image analysis, histological analysis, and the Mankin score. Protein and gene expression was evaluated by enzyme-linked immunosorbent assay, immunohistochemistry, and real-time polymerase chain reaction. MSCs increased production of proinflammatory cytokines under inflammatory conditions. STA21 significantly decreased expression of these proinflammatory molecules via inhibition of STAT3 activity but increased gene expression of molecules related to migration potential and immunomodulation in OA-MSCs. STAT3-inhibited OA-MSCs administrated by IV or IA injection decreased pain severity and cartilage damage in rats with MIA-induced OA rats by decreasing proinflammatory cytokines in the joints. Combined IA and IV-injected STAT3-inhibited OA-MSCs had an additive effect of pain relief in MIA-induced OA rats. STAT3 inhibition may optimize the therapeutic activities of MSCs for treating OA by attenuating pain and progression of MIA by inhibiting inflammation and cartilage damage

The telomere maintenance mechanism spectrum and its dynamics in gliomas

Background : The activation of the telomere maintenance mechanism (TMM) is one of the critical drivers of cancer cell immortality. In gliomas, TERT expression and TERT promoter mutation are considered to reliably indicate telomerase activation, while ATRX mutation and/or loss indicates an alternative lengthening of telomeres (ALT). However, these relationships have not been extensively validated in tumor tissues. Methods : Telomerase repeated amplification protocol (TRAP) and C-circle assays were used to profile and characterize the TMM cross-sectionally (n = 412) and temporally (n = 133) across glioma samples. WES, RNA-seq, and NanoString analyses were performed to identify and validate the genetic characteristics of the TMM groups. Results : We show through the direct measurement of telomerase activity and ALT in a large set of glioma samples that the TMM in glioma cannot be defined solely by the combination of telomerase activity and ALT, regardless of TERT expression, TERT promoter mutation, and ATRX loss. Moreover, we observed that a considerable proportion of gliomas lacked both telomerase activity and ALT. This telomerase activation-negative and ALT negative group exhibited evidence of slow growth potential. By analyzing a set of longitudinal samples from a separate cohort of glioma patients, we discovered that the TMM is not fixed and can change with glioma progression. Conclusions : This study suggests that the TMM is dynamic and reflects the plasticity and oncogenicity of tumor cells. Direct measurement of telomerase enzyme activity and evidence of ALT should be considered when defining TMM. An accurate understanding of the TMM in glioma is expected to provide important information for establishing cancer management strategies.This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF), funded by the Ministry of Science & ICT (NRF-2018M3A9H3021707), and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI21C0239)