Dong Ah Shin

Neuronal Properties, In Vivo Effects, and Pathology of a Huntington's Disease Patient‐Derived Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) generated from somatic cells of patients can be used to model different human diseases. They may also serve as sources of transplantable cells that can be used in novel cell therapies. Here, we analyzed neuronal properties of an iPSC line derived from a patient with a juvenile form of Huntingtons disease (HD) carrying 72 CAG repeats (HD‐iPSC). Although its initial neural inducing activity was lower than that of human embryonic stem cells, we found that HD‐iPSC can give rise to GABAergic striatal neurons, the neuronal cell type that is most susceptible to degeneration in HD. We then transplanted HD‐iPSC‐derived neural precursors into a rat model of HD with a unilateral excitotoxic striatal lesion and observed a significant behavioral recovery in the grafted rats. Interestingly, during our in vitro culture and when the grafts were examined at 12 weeks after transplantation, no aggregate formation was detected. However, when the culture was treated with a proteasome inhibitor (MG132) or when the cells engrafted into neonatal brains were analyzed at 33 weeks, there were clear signs of HD pathology. Taken together, these results indicate that, although HD‐iPSC carrying 72 CAG repeats can form GABAergic neurons and give rise to functional effects in vivo, without showing an overt HD phenotype, it is highly susceptible to proteasome inhibition and develops HD pathology at later stages of transplantation. These unique features of HD‐iPSC will serve as useful tools to study HD pathology and develop novel therapeutics. Stem Cells2012;30:2054–2062

Artificial Disc Replacement Combined With Fusion versus Two-level Fusion in Cervical Two-level Disc Disease

Study Design. A prospective analysis. Objective. The purpose of this study was to compare the clinical and radiologic outcomes of cervical artificial disc replacement (C-ADR) combined with anterior cervical discectomy and fusion (ACDF) and 2-level ACDF in patients with 2-level cervical disc disease. Summary of Background Data. Adjacent segment degeneration is a long-term complication of ACDF, and estimated to affect 25% of patients within 10 years of the initial surgery. Two-level fusion leads to a substantially greater increase in intradiscal pressure than one-level fusion. It has been demonstrated that C-ADR maintains motion at the level of the surgical procedure and decreases strain on the adjacent segments for prevention of adjacent segment degeneration. In the case of 2-level cervical disc disease, hybrid surgery (HS), consisting of C-ADR combined with ACDF, may be a reasonable alternative to 2-level ACDF (2-ACDF). Methods. Between 2004 and 2006, 40 patients undergoing 2-level cervical disc surgery at our hospital were identified who met the following surgical indications: 2 consecutive level degenerative disc disease between C3/4 and C6/7; either a radiculopathy or myelopathy; and no response to conservative treatment for >6 weeks. Twenty patients of the HS group were matched to 20 patients of the 2-ACDF group based on age and gender. Patients were asked to check the neck disability index (NDI) and grade their pain intensity before surgery and at routine postoperative intervals of 1, 3, 6, 12, and 24 months. Dynamic flexion and extension lateral cervical radiographs were obtained in the standing position before surgery and at routine postoperative intervals of 1, 3, 6, 12, and 24 months. The angular range of motion (ROM) for C2–C7 and adjacent segments were measured using the Cobb method with PACS software. Results. The HS group had better NDI recovery 1 and 2 years after surgery (P < 0.05). Postoperative neck pain was less in the HS group 1 month and 1 year after surgery (P < 0.05). There was no difference in arm pain relief between the groups. The HS group showed faster C2–C7 ROM recovery. The mean C2–C7 ROM of the HS group recovered to that of the preoperative value, but that of the 2-ACDF group did not (P < 0.05). The inferior adjacent segment ROM showed significant differences between the groups 6 and 9 months, and 1 and 2 years after surgery (P < 0.05, P < 0.01, P < 0.05, and P < 0.05, respectively). Conclusion. HS is superior to 2-ACDF in terms of better NDI recovery, less postoperative neck pain, faster C2–C7 ROM recovery, and less adjacent ROM increase.

The efficacy of microendoscopic discectomy in reducing iatrogenic muscle injury

OBJECT The objective of this study was to evaluate the invasiveness of microendoscopic discectomy (MED) in comparison with microscopic discectomy (MD) by measuring serum levels of creatine phosphokinase (CPK)-MM and lactate dehydrogenase (LDH)-5, and by comparing visual analog scale (VAS) scores of postoperative pain. METHODS This study included a group of 15 patients who underwent surgery using MED and 15 patients who underwent surgery using MD, both for single-level unilateral herniated nucleus pulposus. The CPK-MM and LDH-5 levels were measured at admission and after 1, 3, and 5 days postoperatively. Pain assessment was recorded using scores raging from 0 to 10 on a subjective VAS at admission and at 1, 3, and 5 days postoperatively. RESULTS The mean CPK-MM levels were lower for the MED group than for the MD group at both 3 (576.1 +/- 286.3 IU/L compared with 968.1 +/- 377.8 IU/L) and 5 days (348.1 +/- 231.0 IU/L compared with 721.7 +/- 463.2) postoperatively (p < 0.05). The mean VAS scores for postoperative back pain were lower in the MED group than in the MD group, both at 1 (3.3 +/- 2.3 compared with 5.8 +/- 1.5) and 5 days (1.9 +/- 1.1 compared with 3.6 +/- 1.1) postoperatively (p < 0.01). CONCLUSIONS The MED procedure is less invasive than MD, and causes less muscle damage and less back pain.

Quantitative proteomic analysis of induced pluripotent stem cells derived from a human Huntington's disease patient.

HD (Huntingtons disease) is a devastating neurodegenerative genetic disorder caused by abnormal expansion of CAG repeats in the HTT (huntingtin) gene. We have recently established two iPSC (induced pluripotent stem cell) lines derived from a HD patient carrying 72 CAG repeats (HD-iPSC). In order to understand the proteomic profiles of HD-iPSCs, we have performed comparative proteomic analysis among normal hESCs (human embryonic stem cells; H9), iPSCs (551-8) and HD-iPSCs at undifferentiated stages, and identified 26 up- and down-regulated proteins. Interestingly, these differentially expressed proteins are known to be involved in different biological processes, such as oxidative stress, programmed cell death and cellular oxygen-associated proteins. Among them, we found that oxidative stress-related proteins, such as SOD1 (superoxide dismutase 1) and Prx (peroxiredoxin) families are particularly affected in HD-iPSCs, implying that HD-iPSCs are highly susceptible to oxidative stress. We also found that BTF3 (basic transcription factor 3) is up-regulated in HD-iPSCs, which leads to the induction of ATM (ataxia telangiectasia mutated), followed by activation of the p53-mediated apoptotic pathway. In addition, we observed that the expression of cytoskeleton-associated proteins was significantly reduced in HD-iPSCs, implying that neuronal differentiation was also affected. Taken together, these results demonstrate that HD-iPSCs can provide a unique cellular disease model system to understand the pathogenesis and neurodegeneration mechanisms in HD, and the identified proteins from the present study may serve as potential targets for developing future HD therapeutics.

Stereotactic spine radiosurgery for intradural and intramedullary metastasis

OBJECT Stereotactic radiosurgery (SRS) has become an important treatment alternative to surgery for a variety of spinal lesions. However, the use of SRS in the management of intradural intramedullary (IDIM) metastasis remains controversial. The aim of this study was to determine the clinical efficacy and safety of SRS for treatment of IDIM metastasis. METHODS Nine patients with 11 IDIM metastases treated with SRS at Henry Ford Hospital were retrospectively reviewed. The mean age at presentation was 50 years, with a range of 14-71 years. There were 4 intradural extramedullary and 7 intramedullary lesions. The radiosurgery procedure used techniques of image-guided and intensitymodulated radiation. The mean treatment dose was 13.8 Gy, with a range of 10-16 Gy. All patients had clinical follow-up (except in 1 lesion), with an emphasis on initial symptoms and ambulatory status, and 8 patients (9 lesions) had imaging studies. The median follow-up duration was 10 months. RESULTS The presenting symptoms were improved in 8 (80%) of 10 evaluable lesions, unchanged in 1 case, and worsened in 1 case. Radiographic responses were seen as follows: complete response in 2 (22%) of 9; partial response in 3 (33%) of 9; stable disease in 3 (33%) of 9; and progressive disease in 1 (11%) of 9. After radiosurgery, 7 patients (78%) remained ambulatory until the last follow-up visit. The overall median survival time after SRS was 8 months, with a range of 2-19 months. No radiation toxicity was detected clinically during the follow-up period. CONCLUSIONS Despite the fact that this was a small series of patients with IDIM metastasis who had limited treatment options, SRS appears to be an effective and safe method of treating patients with these lesions.

Therapeutic potential of human induced pluripotent stem cells in experimental stroke.

Ischemic stroke mainly caused by middle cerebral artery occlusion (MCAo) is a major type of stroke, but there are currently very limited therapeutic options for its cure. Neural stem cells (NSCs) or neural precursor cells (NPCs) derived from various sources are known to survive and improve neurological functions when they are engrafted in animal models of stroke. Induced pluripotent stem cells (iPSCs) generated from somatic cells of patients are novel cells that promise the autologous cell therapy for stroke. In this study, we successfully differentiated iPSCs derived from human fibroblasts into NPCs and found their robust therapeutic potential in a rodent MCAo stroke model. We observed the significant graft-induced behavioral recovery, as well as extensive neural tissue formation. Animal MRI results indicated that the majority of contralaterally transplanted iPSC-derived NPCs migrated to the peri-infarct area, showing a pathotropism critical for tissue recovery. The transplanted animals exhibited the significant reduction of stroke-induced inflammatory response, gliosis and apoptosis, and the contribution to the endogenous neurogenesis. Our results demonstrate that iPSC-derived NPCs are effective cells for the treatment of stroke.

The predisposing factors for the heterotopic ossification after cervical artificial disc replacement

BACKGROUND CONTEXT Heterotopic ossification (HO) is defined as a formation of bone outside the skeletal system. The reported HO occurrence rate in cervical artificial disc replacement (ADR) is unexpectedly high and is known to vary. However, the predisposing factors for HO in cervical ADR have not yet been elucidated. PURPOSE Investigation of the predisposing factors of HO in cervical arthroplasty and the relationship between degeneration of the cervical spine and HO occurrence. STUDY DESIGN Retrospective study to discover predisposing factors of HO in cervical arthroplasty. PATIENT SAMPLE A total of 170 patients who underwent cervical ADR were enrolled including full follow-up clinical and radiologic data. OUTCOME MEASURES Radiologic outcomes were assessed by identification of HOs according to McAfees classifications. METHODS This study enrolled a total of 170 patients who underwent cervical ADR. Pre-existing degenerative change included anterior or posterior osteophytes, ossification of the anterior longitudinal ligament, posterior longitudinal ligament, or ligamentum nuchae. The relationships between basic patient data, pre-existing degenerative change, and HO were investigated using linear logistic regression analysis. RESULTS Among all 170 patients, HO was found in 69 patients (40.6%). Among the postulated predisposing factors, only male gender and artificial disc device type were shown to be statistically significant. Unexpectedly, preoperative degenerative changes in the cervical spine exerted no significant influence on the occurrence of HOs. The odds ratio of male gender compared with female gender was 2.117. With regard to device type, the odds ratios of Mobi-C (LDR medical, Troyes, France) and ProDisc-C (Synthes, Inc., West Chester, PA, USA) were 5.262 and 7.449, respectively, compared with the Bryan disc. CONCLUSIONS Definite differences in occurrence rate according to the gender of patients and the prosthesis type were identified in this study. Moreover, factors indefinably expected to influence HO in the past were not shown to be risk factors thereof, the results of which may be meaningful to future studies.

Effect of Replicated Polymeric Substrate with Lotus Surface Structure on Adipose-Derived Stem Cell Behaviors

We fabricated polystyrene substrates with lotus leaf surface structure (LLSS) and investigated cell behaviors, including attachment, morphology, proliferation, and differentiation of adipose-derived stem cells (ASCs) on them. Compared to the flat substrate, the LLSS substrate induced higher cell attachment rate, but did not significantly change the cell proliferation rate. In addition, ASCs on the LLSS substrate exhibited relatively narrower spreading morphology and less organized cytoskeleton, there by resulting in smaller sizes of cells than those on the flat substrate. According to histochemical staining and RT-PCR analysis, the LLSS substrate induced higher adipogenic differentiation of ASCs than the flat substrate, while chondrogenic and osteogenic differentiation were decreased.

Surgical outcome and prognostic factors of anterior decompression and fusion for cervical compressive myelopathy due to ossification of the posterior longitudinal ligament

BACKGROUND CONTEXT Anterior decompression and fusion (ADF) for ossification of the posterior longitudinal ligament (OPLL) is technically demanding and associated with complications. Although various factors affecting clinical outcome have been investigated in posterior decompression, prognostic factors of ADF remain unclear. PURPOSE The purpose of the study was to identify surgical outcome and prognostic factors of ADF for cervical myelopathy due to OPLL. STUDY DESIGN This was a retrospective case study. PATIENT SAMPLE Between 2005 and 2012, 913 patients underwent decompression surgery for cervical OPLL at our institution. Among them, 131 who underwent ADF and 221 who underwent laminoplasty were enrolled. Inclusion criteria were (1) diagnosis of OPLL; (2) cervical compressive myelopathy; and (3) no trauma, infection, tumor, or previous surgery. We excluded 60 patients with ADF and 157 patients with laminoplasty owing to inadequate follow-up or absence of preoperative myelopathy. Finally, 71 patients with ADF and 64 patients with laminoplasty were enrolled in this study (mean follow-up, 48 vs 41 months). OUTCOME MEASURES Neurologic assessment was conducted using the Japanese Orthopedic Association (JOA) scoring system for cervical myelopathy. Rate of neurologic improvement was calculated by comparing preoperative and postoperative JOA scores. METHODS We investigated the effects of such variables as age, gender, body mass index (BMI), presence of diabetes mellitus (DM), smoking history, type of OPLL, shape of the ossified lesion, occupying ratio of OPLL, presence of intramedullary increased signal intensity (ISI) on magnetic resonance imaging (MRI), and sagittal alignment of the cervical spine on surgical outcome. Severity of ISI was classified into three groups based on T2-weighted sagittal MRI as follows: Grade 0, none; Grade 1, ISI limited to one disc level; or Grade 2, ISI beyond one disc level. This work was supported by the 2013 Korea Health Technology R&D Project of the Ministry of Health and Welfare of the Republic of Korea (A120254). RESULTS In patients with an occupying ratio ≥60% or with presence of ISI on MRI, ADF yielded better surgical outcome than laminoplasty. A higher ISI grade (B=-28.5, p=.000) and a higher occupying ratio (B=0.88, p=.04) were significantly associated with a lower recovery rate (R=0.56, p=.006). Older age also was associated with a lower recovery rate. Gender, BMI, presence of DM, smoking history, type of OPLL, shape of the ossified lesion, and cervical alignment were not associated with recovery rate. CONCLUSION Anterior decompression and fusion has favorable outcome in patients with an occupying ratio ≥60% or with presence of ISI on MRI. Presence of higher ISI grade, higher occupying ratio, and older age were associated with a poor long-term surgical prognosis. Therefore, evaluating ISI and occupying ratio on preoperative MRI is important for selecting the appropriate surgical approach and for predicting clinical outcome after surgery for cervical compressive myelopathy due to OPLL.

Therapeutic effect of BDNF-overexpressing human neural stem cells (HB1.F3.BDNF) in a rodent model of middle cerebral artery occlusion.

Ischemic stroke mainly caused by middle cerebral artery occlusion (MCAo) represents the major type of stroke; however, there are still very limited therapeutic options for the stroke-damaged patients. In this study, we evaluated the neurogenic and therapeutic potentials of human neural stem cells (NSCs) overexpressing brain-derived neurotrophic factor (HB1.F3.BDNF) following transplantation into a rodent model of MCAo. F3.BDNF human NSCs (F3.BDNF) were transplanted into the contralateral side of striatum at 7 days after MCAo, and the transplanted animals were monitored up to 8 weeks using animal MRI and various behavioral tests before they were sacrificed for immunohistochemical analysis. Interestingly, animal MRI results indicate that the majority of contralaterally transplanted neural stem cells were migrated to the peri-infarct area, showing a pathotropism. Transplanted animals exhibited significant behavioral improvements in stepping, rotarod, and modified neurological severity score (mNSS) tests. We also found that the transplanted human cells were colocalized with nestin, DCX, MAP2, DARPP-32, TH, GAD65/67-positive cells, of which results can be correlated with neural regeneration and behavioral recovery in the transplanted animals. More importantly, we were able to detect high levels of human BDNF protein expression, presumably derived from the transplanted F3.BDNF. Taken together, these results provide strong evidence that human neural stem cells (F3.BDNF) are effective in treating stroke animal models.