Ji-Hey Lim

Growth requirements and chromosomal instability of induced pluripotent stem cells generated from adult canine fibroblasts.

In mice and humans, it has been shown that embryonic and adult fibroblasts can be reprogrammed into pluripotency by introducing 4 transcription factors, Oct3/4, Klf4, Sox2, and c-Myc (OKSM). Here, we report the derivation of induced pluripotent stem cells (iPSCs) from adult canine fibroblasts by retroviral OKSM transduction. The isolated canine iPSCs (ciPSCs) were expanded in 3 different culture media [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), or FGF2 plus LIF]. Cells cultured in both FGF2 and LIF expressed pluripotency markers [POU5F1 (OCT4), SOX2, NANOG, and LIN28] and embryonic stem cell (ESC)-specific genes (PODXL, DPPA5, FGF5, REX1, and LAMP1) and showed strong levels of alkaline phosphatase expression. In vitro differentiation by formation of embryoid bodies and by directed differentiation generated cell derivatives of all 3 germ layers as confirmed by mRNA and protein expression. In vivo, the ciPSCs created solid tumors, which failed to reach epithelial structure formation, but expressed markers for all 3 germ layers. Array comparative genomic hybridization and chromosomal fluorescence in situ hybridization analyses revealed that while retroviral transduction per se did not result in significant DNA copy number imbalance, there was evidence for the emergence of low-level aneuploidy during prolonged culture or tumor formation. In summary, we were able to derive ciPSCs from adult fibroblasts by using 4 transcription factors. The isolated iPSCs have similar characteristics to ESCs from other species, but the exact cellular mechanisms behind their unique co-dependency on both FGF2 and LIF are still unknown.

Development of a Model of Sacrocaudal Spinal Cord Injury in Cloned Yucatan MiniPigs for Cellular Transplantation Research

Research into transplantation strategies to treat spinal cord injury (SCI) is frequently performed in rodents, but translation of results to clinical patients can be poor and a large mammalian model of severe SCI is needed. The pig has been considered an optimal model species in which to perform preclinical testing, and the Yucatan minipig can be cloned successfully utilizing somatic cell nuclear transfer (SCNT). However, induction of paralysis in pigs poses significant welfare and nursing challenges. The present study was conducted to determine whether Yucatan SCNT clones could be used to develop an SCI animal model for cellular transplantation research. First, we demonstrated that transection of the sacrocaudal spinal cord in Yucatan SCNT clones produces profound, quantifiable neurological deficits restricted to the tail. We then established that neurospheres could be isolated from brain tissue of green fluorescence protein (GFP) transfected SCNT clones. Finally, we confirmed survival of transplanted GFP-expressing neural stem cells in the SCI lesion and their differentiation into glial and neuronal lineages for up to 4 weeks without immunosuppression. We conclude that this model of sacrocaudal SCI in Yucatan SCNT clones represents a powerful research tool to investigate the effect of cellular transplantation on axonal regeneration and functional recovery.

Gait scoring in dogs with thoracolumbar spinal cord injuries when walking on a treadmill

BackgroundAn inexpensive method of generating continuous data on hind limb function in dogs with spinal cord injury is needed to facilitate multicentre clinical trials. This study aimed to define normal fore limb, hind limb coordination in dogs walking on a treadmill and then to determine whether reliable data could be generated on the frequency of hind limb stepping and the frequency of coordinated stepping in dogs with a wide range of severities of thoracolumbar spinal cord injury.ResultsSixty-nine neurologically normal dogs of different body sizes including seven lame dogs were videotaped walking on the treadmill without prior training and all used the lateral gait of right fore, left hind, left fore, right hind (RF-LH-LF-RH). Severely paraparetic dogs were able to walk on the treadmill for a minimum of 75 seconds, scoring of which generated data representative of function in animals with extremely variable gaits. Fifty consecutive stepping cycles were scored by three observers in 18 dogs with a wide range of disability due to acute thoracolumbar spinal cord injury using a stepping score (hind limb steps/fore limb steps ×100), and a coordination score (coordinated hind limb steps/total hind limb steps ×100). Dogs were also scored using a previously validated ordinal open field score (OFS). Inter- and intraobserver agreement was high as assessed with Cronbach’s alpha test for internal reliability. The stepping and coordination scores were significantly correlated to each other and to the OFS.ConclusionsDogs with naturally occurring spinal cord injury can walk on a treadmill without prior training and their hind limb function can be scored reliably using a stepping score and coordination score. The only requirements for data acquisition are a treadmill and appropriately positioned video camera and so the system can be used in multicentre clinical trials to generate continuous data on neurologic recovery in dogs.

A Placebo-Controlled, Prospective, Randomized Clinical Trial of Polyethylene Glycol and Methylprednisolone Sodium Succinate in Dogs with Intervertebral Disk Herniation

Background Acute intervertebral disk herniation (IVDH) is a common cause of spinal cord injury in dogs and currently there is no proven medical treatment to counter secondary injury effects. Use of methylprednisolone sodium succinate (MPSS) or polyethylene glycol (PEG) as neuroprotectants is advocated but controversial because neither treatment has been tested in placebo‐controlled, randomized, blinded trials in dogs. Hypothesis Polyethylene glycol will improve the outcome of severe spinal cord injury caused by IVDH compared to MPSS or placebo. Animals Client‐owned dogs with acute onset of thoracolumbar IVDH causing paralysis and loss of nociception for <24 hours. Methods Dogs were randomized to receive MPSS, PEG, or placebo; drugs appeared identical and group allocation was masked. Drug administration was initiated once the diagnosis of IVDH was confirmed and all dogs underwent hemilaminectomy. Neurologic function was assessed 2, 4, 8, and 12 weeks postoperatively using an open field gait score (OFS) as the primary outcome measure. Outcomes were compared by the Wilcoxon rank sum test. Results Sixty‐three dogs were recruited and 47.6% recovered ambulation. 17.5% developed progressive myelomalacia but there was no association with group. There was no difference in OFS among groups. Although full study power was not reached, conditional power analyses indicated the futility of continued case recruitment. Conclusions This clinical trial did not show a benefit of either MPSS or PEG in the treatment of acute, severe thoracolumbar IVDH when used as adjunctive medical treatment administered to dogs presenting within 24 hours of onset of paralysis.

Generation and Characterization of Neurospheres from Canine Adipose Tissue-Derived Stromal Cells

Adipose tissue-derived stromal cells (ADSCs) have been identified as a powerful stem cell source for cellular transplantation therapy. The dog is increasingly used as a model of human neurological disease; however, few studies have reported induction of canine ADSCs to neural lineages. We characterized canine ADSCs and investigated whether they could be induced to differentiate into neural lineages. Subcutaneous adipose tissue collected from the dorsal epaxial region of adult dogs aged from 1 to 6 years was cultured to produce ADSCs that were then induced to neural lineages. RT-PCR, flow cytometry, and immunocytochemistry were performed to characterize these cell populations. Morphologically fibroblast-like ADSCs were isolated and had similar characteristics to mesenchymal stem cells. Under neurogenic conditions containing basic fibroblast growth factor and epidermal growth factor, ADSCs formed spherical cellular aggregates that resembled neurospheres. RT-PCR confirmed expression of Sox2 and CD90 by these aggregates. Expression of neural stem/progenitor markers (Nestin, Sox2, Vimentin) and neural lineage markers (A2B5, GFAP, Tuj1) was shown on immunocytochemistry. After differentiation, 60% of the cells were Tuj1 positive. In conclusion, we isolated and generated neural progenitor cells from canine ADSCs. ADSCs have potential for future autologous cell transplantation therapy for neurological disorders.

Alternating Current Electric Fields of Varying Frequencies: Effects on Proliferation and Differentiation of Porcine Neural Progenitor Cells

Application of sinusoidal electric fields (EFs) has been observed to affect cellular processes, including alignment, proliferation, and differentiation. In the present study, we applied low-frequency alternating current (AC) EFs to porcine neural progenitor cells (pNPCs) and investigated the effects on cell patterning, proliferation, and differentiation. pNPCs were grown directly on interdigitated electrodes (IDEs) localizing the EFs to a region accessible visually for fluorescence-based assays. Cultures of pNPCs were exposed to EFs (1 V/cm) of 1 Hz, 10 Hz, and 50 Hz for 3, 7, and 14 days and compared to control cultures. Immunocytochemistry was performed to evaluate the expression of neural markers. pNPCs grew uniformly with no evidence of alignment to the EFs and no change in cell numbers when compared with controls. Nestin expression was shown in all groups at 3 and 7 days, but not at 14 days. NG2 expression was low in all groups. Co-expression of glial fibrillary acidic protein (GFAP) and TUJ1 was significantly higher in the cultures exposed to 10- and 50-Hz EFs than the controls. In summary, sinusoidal AC EFs via IDEs did not alter the alignment and proliferation of pNPCs, but higher frequency stimulation appeared to delay differentiation into mature astrocytes.

The Cutaneous Trunci Muscle Reflex: A Predictor of Recovery in Dogs with Acute Thoracolumbar Myelopathies Caused by Intervertebral Disc Extrusions

OBJECTIVE To determine whether changes in the cutaneous trunci muscle (CTM) reflex are an early predictor of outcome in dogs with severe acute thoracolumbar intervertebral disc extrusion (IVDE). STUDY DESIGN Multicenter prospective cohort study. ANIMALS Dogs (n = 36) with acute IVDE causing paraplegia, loss of nociception in pelvic limbs and tail, and an abnormal CTM reflex postoperatively. METHODS The caudal border of the CTM reflex was established 24 hours after surgery and at discharge, and was reported as moving cranially, caudally, or staying static. Dogs were re-evaluated at 12-20 weeks and at 7-36 months postoperatively. Outcome was classified as improved or unimproved, successful or unsuccessful, and ascending myelomalacia or not, and compared with early movement of the CTM reflex by construction of contingency tables and performing a Fishers exact test. RESULTS By discharge (mean, 4.7 days; SD = 2.10), CTM reflex progression was caudal in 19 dogs, static in 11, and cranial in 6. Five of 6 dogs with cranial movement developed ascending myelomalacia (P < .0001). Seventeen of 19 dogs with caudal movement showed an improvement by 12-20 weeks (P = .0046) and none developed ascending myelomalacia (P = .0013). CONCLUSIONS Postoperative changes of the caudal border of the CTM reflex are an early indicator of outcome in dogs with severe acute IVDE. Cranial movement of the CTM reflex is significantly associated with the development of ascending myelomalacia. Caudal movement is significantly associated with improvement, but not associated with a long-term successful outcome.

Mechanical and Thermal Sensory Testing in Normal Chondrodystrophoid Dogs and Dogs with Spinal Cord Injury caused by Thoracolumbar Intervertebral Disc Herniations

Background Intervertebral disc herniation is a common cause of spinal cord injury (SCI) causing paralysis and sensory loss. Little quantitative information is available on the loss and recovery of sensation in dogs with SCI. Objectives To determine whether quantitative sensory testing (QST) can be used to establish thermal and mechanical sensory thresholds in chrondrodystrophoid dogs and compare thresholds among normal dogs and dogs with different grades of SCI. Animals Thirty‐three client‐owned chondrodystrophoid dogs: 15 normal and 18 SCI dogs. Methods Thermal testing was performed by placing a hot (49°C) and cold (5°C) probe on the dorsal metatarsus and mechanical thresholds were tested using calibrated forceps to apply force to the lateral digit. Stimuli were applied until acknowledged, and response rate, latency, and force applied to response were recorded. Test‐retest repeatability was determined by calculating intraclass correlation coefficients. Response rates were compared using logistic regression and thresholds were compared using Kaplan–Meier Survival curves. Results Testing was feasible with moderate repeatability. Thresholds and response rates were significantly different between normal and SCI dogs for all modalities (P < .001). When dogs were grouped by their clinical grade, each grade was significantly different from normal dogs, and cold stimuli differentiated among all grades. Conclusion and clinical importance Sensory thresholds can be measured reliably in chondrodystrophoid dogs and are altered by SCI. The differences in sensation among neurologic grades indicate that these techniques can be used to further characterize recovery of SCI dogs.

The Sensory Field and Repeatability of the Cutaneous Trunci Muscle Reflex of the Dog

OBJECTIVE To describe the cutaneous trunci muscle (CTM) reflex in dogs. STUDY DESIGN Prospective descriptive study. ANIMALS Normal dogs (n = 155) and 10 dogs with thoracolumbar myelopathies. METHODS The CTM reflex caudal border was assessed from the ilial crests moving cranially until a CTM contraction was elicited. The lateral borders were evaluated at 4 levels and the distance from the midline to the lateral border was expressed asa percentage of the trunk hemicircumference. The caudal border was assessed in 10 dogs with myelopathies by 4 different observers and by 1 observer on 3 occasions;the inter- and intraobserver kappa coefficient was calculated. RESULTS The CTM reflex was elicited in all dogs. Its caudal border was at L5 or L6 in 153 dogs and at L1 and L3 in 2 dogs. The lateral field of the reflex occupied>50%of the hemicircumference of the trunk at each level tested. The mean difference in measurement of the reflex caudal border was 0.55 cm between observers and 0.28 cm for the same observer. The inter- and intraobserver kappa coefficient was 0.67 and 0.87, respectively. CONCLUSIONS The CTM reflex is elicited caudal to L5 in most normal dogs, and the lateral sensory field extends to 50% or more of the circumference of the trunk.Inter- and intraobserver differences in assessment of the caudal border are low.

Evaluation of gene expression and DNA copy number profiles of adipose tissue-derived stromal cells and consecutive neurosphere-like cells generated from dogs with naturally occurring spinal cord injury

OBJECTIVE To evaluate gene expression and DNA copy number in adipose tissue-derived stromal cells (ADSCs) and in ADSC-derived neurosphere-like cell clusters (ADSC-NSCs) generated from tissues of chronically paraplegic dogs. ANIMALS 14 client-owned paraplegic dogs. PROCEDURES Dorsal subcutaneous adipose tissue (< 1 cm3) was collected under general anesthesia; ADSCs were isolated and cultured. Third-passage ADSCs were cultured in neural cell induction medium to generate ADSC-NSCs. Relative gene expression of mesenchymal cell surface marker CD90 and neural progenitor marker nestin was assessed in ADSCs and ADSC-NSCs from 3 dogs by quantitative real-time PCR assay; expression of these and various neural lineage genes was evaluated for the same dogs by reverse transcription PCR assay. Percentages of cells expressing CD90, nestin, glial fibrillary acidic protein (GFAP), and tubulin β 3 class III (TUJ1) proteins were determined by flow cytometry for all dogs. The DNA copy number stability (in samples from 6 dogs) and neural cell differentiation (14 dogs) were assessed with array-comparative genomic hybridization analysis and immunocytochemical evaluation, respectively. RESULTS ADSCs and ADSC-NSCs expressed neural cell progenitor and differentiation markers; GFAP and microtubule-associated protein 2 were expressed by ADSC-NSCs but not ADSCs. Relative gene expression of CD90 and nestin was subjectively higher in ADSC-NSCs than in ADSCs. Percentages of ADSC-NSCs expressing nestin, GFAP, and TUJ1 proteins were substantially higher than those of ADSCs. Cells expressing neuronal and glial markers were generated from ADSC-NSCs and had no DNA copy number instability detectable by the methods used. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested ADSCs can potentially be a safe and clinically relevant autologous source for canine neural progenitor cells. Further research is needed to verify these findings.