Human chemically-derived hepatic progenitors (hCdHs) as a source of liver organoid generation: Application in regenerative medicine, disease modeling, and toxicology testing.

BACKGROUND & AIMS: Several types of human stem cells from embryonic (ESCs) and induced pluripotent (iPSCs) to adult tissue-specific stem cells are commonly used to generate 3D liver organoids for modeling tissue physiology and disease. We have recently established a protocol for direct conversion of primary human hepatocytes (hPHs) from healthy donor livers into bipotent progenitor cells (hCdHs). Here we extended this culture system to generate hCdH-derived liver organoids for diverse biomedical applications. METHODS: To obtain hCdHs, hPHs were cultured in reprogramming medium containing A83-01 and CHIR99021 for 7 days. Liver organoids were established from hCdHs (hCdHOs) and human liver cells (hLOs) using the same donor livers for direct comparison, as well as from hiPSCs. Organoid properties were analyzed by standard in vitro assays. Molecular changes were determined by RT-qPCR and RNA-seq. Clinical relevance was evaluated by transplantation into FRG mice, modeling of alcohol-related liver disease (ARLD), and in vitro drug-toxicity tests. RESULTS: hCdHs were clonally expanded as organoid cultures with low variability between starting hCdH lines. Similar to the hLOs, hCdHOs stably maintained stem cell phenotype based on accepted criteria. However, hCdHOs had an advantage over hLOs in terms of EpCAM expression, efficiency of organoid generation and capacity for directed hepatic differentiation as judged by molecular profiling, albumin secretion, glycogen accumulation, and CYP450 activities. Accordingly, FRG mice transplanted with hCdHOs survived longer than mice injected with hLOs. When exposed to ethanol, hCdHOs developed stronger ARLD phenotype than hLOs as evidenced by transcriptional profiling, lipid accumulation and mitochondrial dysfunction. In drug-induced injury assays in vitro, hCdHOs showed a similar or higher sensitivity response than hPHs. CONCLUSION: hCdHOs provide a novel patient-specific stem cell-based platform for regenerative medicine, toxicology testing and modeling liver diseases.

Rod Fracture Causing Relief of Back Pain That Developed After Adult Lumbar Degenerative Flat-Back Correction Surgery: A Case Report.

A 73-year-old woman underwent deformity correction surgery (anterior lumbar interbody fusion of L2-L3-L4-L5-S1, pedicle subtraction osteotomy at L4, and posterior screw fixation from T10 to the pelvis) due to lumbar degenerative flat-back. Following the operation, the patient experienced pain in her back and buttocks, for which she regularly took medications. She reported frequently feeling a heavy and stretched sensation of pain after the operation in those areas, which made her regret undergoing the operation. However, at 33 months postoperatively, she reported that one day, while getting up from a chair, she felt a crack in her back, which was followed by an improvement in her back and buttock pain; thereafter, she stopped taking pain medications. Follow-up radiography revealed a bilateral rod fracture at the L4-5 level on the right side and at the L3-4 level on the left side. The overall pelvic parameters, except pelvic incidence, slightly changed after the rod fracture. Therefore, the broken rod was replaced and another rod was added to the broken rod area; however, the changed pelvic parameters were not corrected further during the reoperation. Following the reoperation, the patient showed improvements and she no longer required pain medication.

Transforaminal Epiduroscopic Basivertebral Nerve Laser Ablation for Chronic Low Back Pain Associated with Modic Changes: A Preliminary Open-Label Study.

Background: Chronic low back pain (CLBP) arising from degenerative disc disease continues to be a challenging clinical and diagnostic problem whether treated with nonsurgical, pain intervention, or motion-preserving stabilization and arthrodesis. Methods: Fourteen patients with CLBP, greater than 6 months, unresponsive to at least 4 months of conservative care were enrolled. All patients were treated successfully following screening using MRI findings of Modic type I or II changes and positive confirmatory provocative discography to determine the affected levels. All patients underwent ablation of the basivertebral nerve (BVN) using 1414 nm Nd:YAG laser-assisted energy guided in a transforaminal epiduroscopic approach. Macnab's criteria and visual analog scale (VAS) score were collected retrospectively at each follow-up interval. Results: The mean age was 46 ± 9.95 years. The mean symptoms duration was 21.21 ± 21.87 months. The mean follow-up was 15.3 ± 2.67 months. The preoperative VAS score of 7.79 ± 0.97 changed to 1.92 ± 1.38, postoperatively (P < 0.01). As per Macnab's criteria, seven patients (50%) had excellent, six patients (42.85%) had good, and one patient (7.14%) had fair outcomes. Conclusion: The transforaminal epiduroscopic basivertebral nerve laser ablation (TEBLA) appears to be a promising option in carefully selected patients with CLBP associated with the Modic changes.

Full Endoscopic Lumbar Discectomy using the Calcification Floating Technique for Symptomatic Partially Calcified Lumbar Herniated Nucleus Pulposus.

BACKGROUND: Partially calcified lumbar herniated nucleus pulposus (HNP) can cause severe radiating pain and neurologic symptoms requiring surgical treatment. As it is not safe to enforce conventional endoscopic lumbar discectomy using trephine or burr to remove the partially calcified disc, we report a calcification floating technique using a working channel for the treatment of these cases. METHODS: We retrospectively analyzed 31 patients who underwent full endoscopic discectomy using this technique for partially calcified lumbar HNP between April 2009 and June 2013. Calcification floating technique was performed by inserting the working channel around the partially calcified HNP and then rotating the working channel around it to remove the lesion. We analyzed the outcomes with a Visual Analogue Scale (VAS), Oswestry Disability Index (ODI), and complication rate. RESULTS: The mean follow-up period was 26.58 ± 11.2 months. The interlaminar approach was used in 15 cases, and the transforaminal approach was used in 16 cases. The mean VAS of 8.19 ± 0.65 before surgery was decreased to 1.29 ± 0.69 at the last follow-up. The mean ODI score before surgery was decreased at the last follow-up, from 41.32 ± 2.87 to 9.87 ± 3.47. Mean operative duration was 45 ± 12 minutes per level. None of the patients required revision surgery or developed any major complication. CONCLUSIONS: Calcification floating technique is a safe and effective method for the treatment of partially calcified lumbar HNP.

Functional Limitations Due to Stiffness After Long-Level Spinal Instrumented Fusion Surgery to Correct Lumbar Degenerative Flat Back.

STUDY DESIGN: A retrospective analysis of functional limitations due to stiffness after long-level spinal instrumented fusion surgery to correct lumbar degenerative flat back was performed. OBJECTIVE: To analysis the functional limitations in patients treated surgically for adult lumbar degenerative flat back (ALDFB) with long-level instrumented fusion to the sacrum or pelvis. SUMMARY OF BACKGROUND DATA: Long-level instrumented fusion for ALDFB decreases back pain and spinal deformity. On the contrary, this surgery considerably eliminates spinal range of motion. This may have the potential to impair function and ability to perform activities of daily living (ADLs). METHODS: Consecutive 44 patients who underwent long-level instrumented fusion to the sacrum or pelvis for ALDFB were retrospectively included in this study. All patients were followed up for a minimum of 13 months. The Lumbar Stiffness Disability Index for Korean Lifestyle and Oswestry Disability Index were administered and analyzed to assess the impact of spinal stiffness on daily living. Cohorts were defined based on the upper instrumented vertebrae (above T10 [group 1] or below L1 [group 2]) and lower instrumented vertebrae (S1 pedicle screw [group S] or iliac bolt screw [group I]). RESULTS: All patients showed deteriorated postoperative ADLs compared to preoperative values. Group 1 showed deterioration postoperatively compared to group 2. Group 1 showed deteriorated postoperative ADLs compared to preoperative values. In group 2, question 5 and 7 showed deterioration postoperatively compared to preoperative values, and question 2 and 10 showed improvement postoperatively compared to preoperative values. Group I showed deterioration postoperatively compared to group S. CONCLUSION: This study will hopefully allow surgeons to provide patients with ALDFB with a more informed explanation of expected surgery effects on specific ADLs. LEVEL OF EVIDENCE: 3.

Lumbar chronic subdural hematoma mimicking an intradural extramedullary tumor: A case report.

BACKGROUND: Chronic spinal subdural hematomas are extremely rare with only 28 cases reported in the literature. Nevertheless, they should be considered among the differential diagnoses for spinal intradural/extramedullary lesions. CASE REPORT: A 65-year-old male presented with progressive back pain and right S1 radiculopathy. Magnetic resonance imaging scan revealed a right-sided posterolateral intradural/extramedullary lesion at the L5-S1 level. It was hyperintense on T1 and hypointense on T2-weighted images; on the short TI inversion recovery sequence it was hyperintense. The lesion was excised through a right L5 hemilaminectomy, and the patient was neurologically intact postoperatively. Histopathology revealed a chronic subdural hematoma. CONCLUSION: Chronic spinal subdural hematoma can mimic intradural extramedullary spinal tumors even in the absence of trauma and/or coagulopathies.

Regulation of dendritic arborization by BCR Rac1 GTPase-activating protein, a substrate of PTPRT.

Dendritic arborization is important for neuronal development as well as the formation of neural circuits. Rac1 is a member of the Rho GTPase family that serve as regulators of neuronal development. Breakpoint cluster region protein (BCR) is a Rac1 GTPase-activating protein that is abundantly expressed in the central nervous system. Here, we show that BCR plays a key role in neuronal development. Dendritic arborization and actin polymerization were attenuated by overexpression of BCR in hippocampal neurons. Knockdown of BCR using specific shRNAs increased the dendritic arborization as well as actin polymerization. The number of dendrites in null mutant BCR(-/-) mice was considerably increased compared with that in wild-type mice. We found that the function of the BCR GTPase-activating domain could be modulated by protein tyrosine phosphatase receptor T (PTPRT), which is expressed principally in the brain. We demonstrate that tyrosine 177 of BCR was the main target of PTPRT and the BCR mutant mimicking dephosphorylation of tyrosine 177 alleviated the attenuation of dendritic arborization. Additionally the attenuated dendritic arborization found upon BCR overexpression was relieved upon co-expression of PTPRT. When PTPRT was knocked down by a specific shRNA, the dendritic arborization was significantly reduced. The activity of the BCR GTPase-activating domain was modulated by means of conversions between the intra- and inter-molecular interactions, which are finely regulated through the dephosphorylation of a specific tyrosine residue by PTPRT. We thus show conclusively that BCR is a novel substrate of PTPRT and that BCR is involved in the regulation of neuronal development via control of the BCR GTPase-activating domain function by PTPRT.

Regulation of synaptic Rac1 activity, long-term potentiation maintenance, and learning and memory by BCR and ABR Rac GTPase-activating proteins.

Rho family small GTPases are important regulators of neuronal development. Defective Rho regulation causes nervous system dysfunctions including mental retardation and Alzheimer's disease. Rac1, a member of the Rho family, regulates dendritic spines and excitatory synapses, but relatively little is known about how synaptic Rac1 is negatively regulated. Breakpoint cluster region (BCR) is a Rac GTPase-activating protein known to form a fusion protein with the c-Abl tyrosine kinase in Philadelphia chromosome-positive chronic myelogenous leukemia. Despite the fact that BCR mRNAs are abundantly expressed in the brain, the neural functions of BCR protein have remained obscure. We report here that BCR and its close relative active BCR-related (ABR) localize at excitatory synapses and directly interact with PSD-95, an abundant postsynaptic scaffolding protein. Mice deficient for BCR or ABR show enhanced basal Rac1 activity but only a small increase in spine density. Importantly, mice lacking BCR or ABR exhibit a marked decrease in the maintenance, but not induction, of long-term potentiation, and show impaired spatial and object recognition memory. These results suggest that BCR and ABR have novel roles in the regulation of synaptic Rac1 signaling, synaptic plasticity, and learning and memory, and that excessive Rac1 activity negatively affects synaptic and cognitive functions.

Phosphorylation of stargazin by protein kinase A regulates its interaction with PSD-95.

Stargazin is the first transmembrane protein known to associate with AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) glutamate receptors (AMPARs) and regulate their synaptic targeting by two distinct mechanisms, specifically via delivery of AMPARs to the surface membrane and synaptic targeting of these receptors by binding to PSD-95/SAP-90 and related PDZ proteins. However, it is not known whether and how this stargazin-mediated synaptic targeting of AMPARs is regulated. Stargazin interacts with the PDZ domains of PSD-95 through the C-terminal PDZ-binding motif. The stargazin C terminus contains a consensus sequence for phosphorylation by cAMP-dependent protein kinase A (PKA). Phosphorylation site-specific stargazin antibodies reveal that the stargazin C terminus is phosphorylated at the Thr-321 residue in heterologous cells and in vivo. Stargazin phosphorylation is enhanced by the catalytic subunit of PKA. Mutations mimicking stargazin phosphorylation (T321E and T321D) lead to elimination of yeast two-hybrid interactions, in vitro coimmunoprecipitation, and coclustering between stargazin and PSD-95. Phosphorylated stargazin shows a selective loss of coimmunoprecipitation with PSD-95 in heterologous cells and limited enrichment in postsynaptic density fractions of rat brain. These results suggest that phosphorylation of the stargazin C terminus by PKA regulates its interaction with PSD-95 and synaptic targeting of AMPARs.