Secondary Pulmonary Alveolar Proteinosis Development during the Treatment for Anti-aminoacyl-tRNA Synthetase Antibody-positive Interstitial Lung Disease.

A 70-year-old woman with anti-aminoacyl-tRNA synthetase (ARS) antibody-positive interstitial lung disease (ARS-ILD) received daily medications and regular cyclophosphamide cycles for recurring exacerbations. Approximately four years after immunosuppression initiation, the patient was admitted for progressive dyspnea on exertion. Chest computed tomography (CT) findings were suggestive of acute exacerbation. Despite intensified immunosuppressive treatment, the radiographic findings worsened, and serum Krebs von den Lungen-6 (KL-6) levels increased. A bronchoalveolar lavage fluid (BALF) examination revealed amorphous globules and alveolar macrophages with eosinophilic granules. Owing to negative anti-GM-CSF antibody tests, a diagnosis of secondary pulmonary alveolar proteinosis (PAP) was established.

Large-vessel vasculitis induced by pegfilgrastim and immune checkpoint inhibitor in a patient with small-cell lung cancer.

A 75-year-old woman with stage IVB (cT3N3M1c) extensive disease small-cell lung cancer was treated with carboplatin, etoposide, and atezolizumab. Ten days after pegfilgrastim initiation, during the second chemotherapy cycle, she experienced back pain. Contrast-enhanced computed tomography revealed soft tissue thickening around the descending aorta and brachiocephalic artery. She was diagnosed with atezolizumab and pegfilgrastim-induced large-vessel vasculitis (LVV) and was treated with prednisolone, which was tapered and discontinued after 14 weeks, with no symptom recurrence. LVV should be included in the differential diagnosis of patients with nonspecific body pain when pegfilgrastim and immune checkpoint inhibitors are used in combination.

Lysine demethylase 2B regulates angiogenesis via Jumonji C dependent suppression of angiogenic transcription factors.

Vascular endothelial growth factor (VEGF) signaling plays a central role in vascular development and maintenance of vascular homeostasis. In endothelial cells (ECs), VEGF activates the gene expression of angiogenic transcription factors (TFs), followed by induction of downstream angiogenic responsive genes. Recent findings support that histone modification dynamics contribute to the transcriptional control of genes that are important for EC functions. Lysine demethylase 2B (KDM2B) demethylates histone H3K4me3 and H3K36me2/3 and mediates the monoubiquitination of histone H2AK119. KDM2B functions as a transcriptional repressor in somatic cell reprogramming and tumor development. However, the role of KDM2B in VEGF signaling remains to be elucidated. Here, we show that KDM2B knockdown enhances VEGF-induced angiogenesis in cultured human ECs via increased migration and proliferation. In contrast, ectopic expression of KDM2B inhibits angiogenesis. The function of KDM2B may depend on its catalytic Jumonji C domain. Genome-wide analysis further reveals that KDM2B selectively controls the transcription of VEGF-induced angiogenic TFs that are associated with increased H3K4me3/H3K36me3 and decreased H2AK119ub. These findings suggest an essential role of KDM2B in VEGF signaling in ECs. As dysregulation of VEGF signaling in ECs is involved in various diseases, including cancer, KDM2B may be a potential therapeutic target in VEGF-mediated vasculopathic diseases.

Protocol for analysis of integrin-mediated cell adhesion of lateral plate mesoderm cells isolated from zebrafish embryos.

Lateral plate mesoderm (LPM) cells differentiate into various cell types including endothelial and hematopoietic cells. In zebrafish embryos, LPM cells migrate toward the midline along the ventral surfaces of somites during which their cell fate specification depends upon efficient integrin-mediated cell adhesion and migration. Herein, we present a protocol for analysis of integrin-mediated cell adhesion of LPM cells isolated from zebrafish embryos. This allows the study of the molecular mechanisms underlying integrin activation required for LPM cell fate specification. For complete details on the use and execution of this protocol, please refer to Rho et al. (2019).

Interleukin-11-expressing fibroblasts have a unique gene signature correlated with poor prognosis of colorectal cancer.

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.

Preclinical Characterization of the Radioimmunoconjugate 111In or 90Y-FF-21101 Against a P-Cadherin-Expressing Tumor in a Mouse Xenograft Model and a Nonhuman Primate.

P-cadherin is overexpressed in various cancers and can be a target for radioimmunotherapy. We investigated the preclinical pharmacokinetics and pharmacology of FF-21101, an 111In- or 90Y-conjugated monoclonal antibody against P-cadherin, to evaluate its clinical applications. Methods: The radiochemical purity, binding affinity, and in vitro serum stability of 111In or 90Y-labeled FF-21101 were evaluated. The pharmacokinetics of 111In or 90Y-FF-21101 were compared in normal mice. Tumor accumulation after 111In-FF-21101 administration was investigated in mice bearing subcutaneous tumors with high (NCI-H1373), moderate (EBC-1), or no (A549) P-cadherin expression. The tumor suppression effect after a single intravenous injection of 90Y-FF-21101 was assessed in NCI-H1373 and EBC-1 mouse xenograft models. The relationship between antibody dose and tumor accumulation was investigated in the NCI-H1373 mouse xenograft model. The absorbed radiation dose in humans after injection of 90Y-FF-21101 was estimated using γ-camera images of cynomolgus monkeys. Results: The radiochemical purities of 111In- and 90Y-FF-21101 were 98.2% ± 2.5% (n = 9) and 99.3% ± 0.6% (n = 5), respectively. The dissociation constants were 1.083 nM for 111In-FF-21101 and 1.367 nM for 90Y-FF-21101. Both 111In- and 90Y-FF-21101 were stable in human serum after 96 h of incubation and exhibited similar pharmacokinetics in normal mice. The tumor accumulation of 111In-FF-21101 was closely related to the intensity of P-cadherin expression in the cells. 90Y-FF-21101 showed significant tumor growth inhibition, indicating that NCI-H1373 and EBC-1 recurrence was not observed after intravenous administration of 3.7 and 7.4 MBq, respectively of 90Y-FF-21101 per animal. Tumor uptake in the mouse xenograft model and estimated absorbed radiation doses in the spleen of monkeys decreased with increasing antibody doses of 111In-FF-21101. Conversely, the estimated absorbed radiation dose in the red marrow increased with increasing antibody dose. An antibody dose of 4.8 mg/m2 was considered appropriate for humans, on the basis of efficacy and safety. The maximum tolerated administered activity of 90Y-FF-21101 was estimated to be 2,886 MBq/human. Conclusion: FF-21101 radioimmunotherapy exhibited high antitumor affinity and antitumor efficacy in mouse xenograft models. Extrapolation of the pharmacokinetics in monkeys to humans suggests the potential for clinical application of FF-21101 for treating P-cadherin-expressing tumor.

Phase I Study of P-cadherin-targeted Radioimmunotherapy with 90Y-FF-21101 Monoclonal Antibody in Solid Tumors.

PURPOSE: 90Y-FF-21101 is an Yttrium-90-conjugated, chimeric mAb that is highly specific for binding to human placental (P)-cadherin, a cell-to-cell adhesion molecule overexpressed and associated with cancer invasion and metastatic dissemination in many cancer types. We report the clinical activity of 90Y-FF-21101 in a first-in-human phase I study in patients with advanced solid tumors. PATIENTS AND METHODS: The safety and efficacy of 90Y-FF-21101 were evaluated in a phase I 3+3 dose-escalation study in patients with advanced solid tumors (n = 15) over a dose range of 5-25 mCi/m2. Dosimetry using 111In-FF-21101 was performed 1 week prior to assess radiation doses to critical organs. Patients who demonstrated clinical benefit received repeated 90Y-FF-21101 administration every 4 months. RESULTS: 111In-FF-21101 uptake was observed primarily in the spleen, kidneys, testes, lungs, and liver, with tumor uptake observed in the majority of patients. Organ dose estimates for all patients were below applicable limits. P-cadherin expression H-scores ranged from 0 to 242 with 40% of samples exhibiting scores ≥100. FF-21101 protein pharmacokinetics were linear with increasing antibody dose, and the mean half-life was 69.7 (±12.1) hours. Radioactivity clearance paralleled antibody clearance. A complete clinical response was observed in a patient with clear cell ovarian carcinoma, correlating with a high tumor P-cadherin expression. Stable disease was observed in a variety of other tumor types, without dose-limiting toxicity. CONCLUSIONS: The favorable safety profile and initial antitumor activity observed for 90Y-FF-21101 warrant further evaluation of this radioimmunotherapeutic (RIT) approach and provide initial clinical data supporting P-cadherin as a potential target for cancer treatment.

Hlf marks the developmental pathway for hematopoietic stem cells but not for erythro-myeloid progenitors.

Before the emergence of hematopoietic stem cells (HSCs), lineage-restricted progenitors, such as erythro-myeloid progenitors (EMPs), are detected in the embryo or in pluripotent stem cell cultures in vitro. Although both HSCs and EMPs are derived from hemogenic endothelium, it remains unclear how and when these two developmental programs are segregated during ontogeny. Here, we show that hepatic leukemia factor (Hlf) expression specifically marks a developmental continuum between HSC precursors and HSCs. Using the Hlf-tdTomato reporter mouse, we found that Hlf is expressed in intra-aortic hematopoietic clusters and fetal liver HSCs. In contrast, EMPs and yolk sac hematopoietic clusters before embryonic day 9.5 do not express Hlf HSC specification, regulated by the Evi-1/Hlf axis, is activated only within Hlf+ nascent hematopoietic clusters. These results strongly suggest that HSCs and EMPs are generated from distinct cohorts of hemogenic endothelium. Selective induction of the Hlf+ lineage pathway may lead to the in vitro generation of HSCs from pluripotent stem cells.

Rap1b Promotes Notch-Signal-Mediated Hematopoietic Stem Cell Development by Enhancing Integrin-Mediated Cell Adhesion.

Hematopoietic stem cells (HSCs) emerge from hemogenic endothelium (HE) within the ventral portion of the dorsal aorta during vertebrate development. In zebrafish, Notch signaling induces HE specification from posterior lateral plate mesoderm (PLPM) cells as they migrate over the ventral surface of the somite. During migration, PLPM cells make close contact with Notch-ligand-expressing somitic cells to acquire HE identity. Herein, we show in zebrafish that the small GTPase Rap1b regulates HSC development by potentiating Notch-mediated HE specification. PLPM cells migrate toward the midline along the somite boundary where fibronectin accumulates. Rap1b stimulates integrin ß1 to enhance PLPM cell adhesion to fibronectin localized at the somite boundary. Rap1b-induced integrin-ß1-mediated adhesion to fibronectin leads to the spreading of PLPM cells to facilitate their physical contact with the Notch-ligand-expressing somitic cells, thereby promoting Notch-mediated HE specification. Thus, we have revealed an unexpected role of Rap1-induced integrin-mediated cell adhesion in HSC development.

Induced Pluripotent Stem Cells Reprogrammed with Three Inhibitors Show Accelerated Differentiation Potentials with High Levels of 2-Cell Stage Marker Expression.

Although pluripotent stem cells can generate various types of differentiated cells, it is unclear why lineage-committed stem/progenitor cells derived from pluripotent stem cells are decelerated and why the differentiation-resistant propensity of embryonic stem cell (ESC)/induced pluripotent stem cell (iPSC)-derived cells is predominant compared with the in vivo equivalents derived from embryonic/adult tissues. In this study, we demonstrated that iPSCs reprogrammed and maintained with three chemical inhibitors of the fibroblast growth factor 4-mitogen-activated protein kinase cascade and GSK3ß (3i) could be differentiated into all three germ layers more efficiently than the iPSCs reprogrammed without the 3i chemicals, even though they were maintained with 3i chemicals once they were reprogrammed. Although the iPSCs reprogrammed with 3i had increased numbers of Zscan4-positive cells, the Zscan4-positive cells among iPSCs that were reprogrammed without 3i did not have an accelerated differentiation ability. These observations suggest that 3i exposure during the reprogramming period determines the accelerated differentiation/maturation potentials of iPSCs that are stably maintained at the distinct state.

Compromised vitality of spermatozoa after contact with colonic mucosa in mice: implications for fertility in colon vaginoplasty patients.

AIM OF THE STUDY: Colon vaginoplasty (CV) is often performed for cloacal malformation (CM). We used mice to study the vitality of spermatozoa after contact with colonic mucosa as a factor contributing to infertility. METHODS: Spermatozoa isolated from the epididymides of C57BL/6J male mice (n = 23) were syringed directly into the vaginas (Vag-group) or colons (Colo-group) of female mice (n = 45). Vitality was determined by assessing motility using computer-assisted sperm analysis, viability by staining with SYBR-14 and propidium iodide, and fertility by in vitro fertilization, prior to deposition, and at 5, 10, 30, and 60 min after deposition. MAIN RESULTS: Motility was significantly decreased in Colo only at 10 and 60 min. Viability of Colo spermatozoa was significant at all assessment times, except at 10 min. Normal fertilization was observed with all Vag spermatozoa, but with Colo, there was arrest of embryo development with spermatozoa collected at 5 and 10 min, and no fertilization with spermatozoa collected at 30 and 60 min. CONCLUSIONS: The vitality of spermatozoa is compromised by contact with colonic mucosa which could contribute to infertility in CM after CV, because their ovaries and fallopian tubes are considered to be normal.

Vasoinhibin, an N-terminal Prolactin Fragment, Directly Inhibits Cardiac Angiogenesis in Three-dimensional Heart Culture.

Vasoinhibins (Vi) are fragments of the growth hormone/prolactin (PRL) family and have antiangiogenic functions in many species. It is considered that Vi derived from PRL are involved in the pathogenesis of peripartum cardiomyopathy (PPCM). However, the pathogenic mechanism of PPCM, as well as heart angiogenesis, is not yet clear. Therefore, the aim of the present study is to clarify whether Vi act directly on angiogenesis inhibition in heart blood vessels. Endothelial cell viability was decreased by Vi treatment in a culture experiment. Furthermore, expression of proangiogenic genes, such as vascular endothelial growth factor, endothelial nitric oxide synthase, and VE-cadherin, were decreased. On the other hand, apoptotic factor gene, caspase 3, and inflammatory factor genes, tumor necrosis factor α and interleukin 6, were increased by Vi treatment. In three-dimensional left ventricular wall angiogenesis assay in mice, Vi treatment also inhibited cell migration, neovessel sprouting, and growth toward collagen gel. These data demonstrate that Vi treatment directly suppresses angiogenesis of the heart and support the hypothesis that Vi induce PPCM.

Laparoscopic Davydov Procedure for the Creation of a Neovagina in Patients with Mayer-Rokitansky-Kuster-Hauser Syndrome: Analysis of 7 Cases.

OBJECTIVE: Several surgical techniques have been described for creating a neovagina in patients with Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome, however as yet there is no standardized treatment. The aim of this report is to assess anatomic and functional outcomes after the laparoscopic Davydov procedure for the creation of a neovagina in patients with MRKH syndrome. METHODS: Seven patients with MRKH syndrome underwent the laparoscopic Davydov technique from January 2005 to August 2010. The anatomic and functional results were evaluated after 3, 6, 12, 24, 36, 48, and 60 months. RESULTS: The surgical procedure was performed with no major complications except in one case in which an intraoperative bladder injury occurred and was successfully corrected. The mean duration of surgery was 162.9 minutes (range, 120-230 min). Mean lengths/widths (cm) of the neovagina were 6.4/2.6, 6.5/2.5, 6.5/2.8, 6.4/2.8, 7.1/2.8, and 7.2/2.8 at 3, 6,12, 24, 36, 48, and 60 postoperative months, respectively. CONCLUSION: The laparoscopic Davydov procedure seems to be a safe and effective surgical treatment for patients with MRKH syndrome if postoperative intermittent self dilation was done.

Endoscopic detection of the gastric lesions of peripheral T-cell lymphoma.

An 82-year-old Japanese man presented with a gastric involvement of peripheral T-cell lymphoma, not otherwise specified. Although gastrointestinal lesions were not detected on computed tomography, oesophagogastroduodenoscopy revealed a slight elevation of the gastric mucosa, with changes in mucosal colour and the presence of abnormal microvessels. This led to the prompt detection of gastric involvement in lymphoma. This case highlights the usefulness of detailed observation of the gastric mucosa for the endoscopic detection of gastric involvement of peripheral T-cell lymphoma.

Aberrant differentiation of Tsc2-deficient teratomas associated with activation of the mTORC1-TFE3 pathway.

The model animal of renal cell carcinoma (RCC), the Eker rat, has a germline mutation in the tuberous sclerosis 2 (Tsc2) gene. Heterozygous mutants develop RCCs by second hit in the wild-type Tsc2 allele, whereas homozygous mutants are embryonic lethal. In the present study, a new cell differentiation model was developed to study the mechanism of Tsc2 mutation-associated pathogenesis by generating Tsc2-deficient embryonic stem cells (ESCs) from Eker rats. Tsc2+/+, Tsc2+/- and Tsc2-/- ESCs were all capable of generating three germ layers: mesoderm, ectoderm, and endoderm. Interestingly, epithelial tumor-like abnormal ductal structures were reproducibly observed in Tsc2-/- teratomas from different ESC lines. Immunohistochemical analysis revealed that mammalian target of rapamycin complex 1 (mTORC1) signaling was activated in abnormal ducts of Tsc2-/- teratomas, on the basis of positive staining for p-S6 and p-4EBP1. In these abnormal ducts, expression levels of epithelial markers (i.e., megalin and cubilin) and the cytoplasmic localization of E-cadherin and ß-catenin were similar to those in Eker rat RCCs. Moreover, a transcription factor regulated by mTORC1, named TFE3, was located in the nuclei of abnormal ducts and Eker rat RCCs. As a negative regulator of ESC differentiation, TFE3 may result in tissue-specific differentiation defects related to tumorigenesis in Eker rats and Tsc2-/- teratomas. The present study suggests that ESCs derived from Eker rats constitute a novel experimental tool with which to analyze differentiation defects and cell-type specific pathogenesis associated with Tsc2 deficiency.

A paraganglioma in a hypertensive patient with unilateral renal hypoplasia.

We report the case of a 46-year-old hypertensive Japanese female with renal insufficiency related to unilateral renal hypoplasia. The patient was found to have developed paraganglioma in the retroperitoneal space over a 5-year period. Catecholamine-producing tumors are not usually recognized as conditions associated with renal hypoplasia. Our long-term observation of the patient eventually led us to the diagnosis of paraganglioma. In hypertensive patients with chronic kidney disease, not only the renin-angiotensin-aldosterone system but also catecholamine activity may be involved, particularly in the patients whose cases are complicated with unilateral renal hypoplasia.

Complication of chronic eosinophilic pneumonia in an elderly patient with Sjögren syndrome.

An 81-year-old Japanese male with primary Sjögren syndrome (pSS) developed a low-grade fever and productive cough which were refractory to antibiotic therapy. Based on the high level of eosinophils observed in his bronchial alveolar lavage, he was diagnosed with chronic eosinophilic pneumonia (CEP) and successfully treated by oral prednisolone. Interstitial lung diseases associated with pSS (pSS-ILDs) usually present as nonspecific interstitial pneumonia or usual interstitial pneumonia; therefore, the present case is extremely unique in that the patient's condition was complicated with CEP. A diagnosis of advanced gallbladder cancer was made in the patient's clinical course, suggesting the advisability of a whole-body workup in cases of pSS, especially in elderly patients.

Mutual effects of melatonin and activin on induction of aldosterone production by human adrenocortical cells.

Melatonin has been reported to suppress adrenocorticotropin (ACTH) secretion in the anterior pituitary and cortisol production in the adrenal by different mechanisms. However, the effect of melatonin on aldosterone production has remained unknown. In this study, we investigated the role of melatonin in the regulation of aldosterone production using human adrenocortical H295R cells by focusing on the activin system expressed in the adrenal. Melatonin receptor MT1 mRNA and protein were expressed in H295R cells and the expression levels of MT1 were increased by activin treatment. Activin increased ACTH-induced, but not angiotensin II (Ang II)-induced, aldosterone production. Melatonin alone did not affect basal synthesis of either aldosterone or cortisol. However, melatonin effectively enhanced aldosterone production induced by co-treatment with ACTH and activin, although melatonin had no effect on aldosterone production induced by Ang II in combination with activin. These changes in steroidogenesis became apparent when the steroid production was evaluated by the ratio of aldosterone/cortisol. Melatonin also enhanced dibutyryl-AMP-induced aldosterone/cortisol levels in the presence of activin, suggesting a functional link to the cAMP-PKA pathway for induction of aldosterone production by melatonin and activin. In accordance with the data for steroids, ACTH-induced, but not Ang II-induced, cAMP synthesis was also amplified by co-treatment with melatonin and activin. Furthermore, the ratio of ACTH-induced mRNA level of CYP11B2 compared with that of CYP17 was amplified in the condition of treatment with both melatonin and activin. In addition, melatonin increased expression of the activin type-I receptor ALK-4 but suppressed expression of inhibitory Smads6/7, leading to the enhancement of Smad2 phosphorylation. Collectively, the results showed that melatonin facilitated aldosterone production induced by ACTH and activin via the cAMP-PKA pathway. The results also suggested that mutual enhancement of melatonin and activin receptor signaling is involved in the induction of aldosterone output by adrenocortical cells.

Action-at-a-distance mutagenesis induced by oxidized guanine in Werner syndrome protein-reduced human cells.

8-Oxo-7,8-dihydroguanine (G(O), 8-hydroxyguanine) in DNA is one of the most important oxidatively damaged bases and causes G:C → T:A substitution mutations. The Werner syndrome protein (WRN) is a cancer-related RecQ DNA helicase and plays many roles in DNA replication and repair. To examine the relationships between G(O)-induced mutations and WRN, shuttle plasmid DNA containing a G(O):C pair in the supF gene was transfected into human U2OS cells, in which WRN was knocked down. The plasmid DNA replicated in the knockdown cells was introduced into an Escherichia coli indicator strain. The knockdown of WRN increased the mutant frequency of the G(O)-plasmid DNA. Unexpectedly, however, the WRN knockdown only slightly enhanced the targeted G:C → T:A mutation. Instead, base-substitution mutations at various positions were more frequently detected, with statistical significance. The results obtained in this study suggested that the reduction of the cancer-related WRN induced action-at-a-distance mutagenesis by the G(O):C pair in human cells. In addition, the WRN knockdown decreased the G(O):A-induced A:T → C:G mutations, suggesting that WRN may enhance the mutations caused by G(O) in the nucleotide pool.

Establishment of Tsc2­deficient rat embryonic stem cells.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by TSC1 or TSC2 mutations. TSC causes the development of tumors in various organs such as the brain, skin, kidney, lung, and heart. The protein complex TSC1/2 has been reported to have an inhibitory function on mammalian target of rapamycin complex 1 (mTORC1). Treatment with mammalian target of rapamycin (mTOR) inhibitors has demonstrated tumor­reducing effects in patients with TSC but is also associated with various adverse effects. In recent years, experiments involving in vivo differentiation of pluripotent stem cells have been reported as useful in elucidating mechanisms of pathogenesis and discovering new therapeutic targets for several diseases. To reveal the molecular basis of the pathogenesis caused by the Tsc2 mutation, we derived embryonic stem cells (ESCs) from Eker rats, which have the Tsc2 mutation and develop brain lesions and renal tumors. Although several studies have reported the necessity of Tsc1 and Tsc2 regulation to maintain ESCs and hematopoietic stem cells, we successfully established not only Tsc2+/+ and Tsc2+/- ESCs but also Tsc2-/- ESCs. We confirmed that these cells express pluripotency markers and retain the ability to differentiate into all three germ layers. Comprehensive gene expression analysis of Tsc2+/+ and Tsc2+/- ESCs revealed similar profiles, whereas the profile of Tsc2-/- ESCs was distinct from these two. In vitro differentiation experiments using these ESCs combined with in vivo experiments may reveal the mechanism of the tissue­specific pathogenesis caused by the Tsc2 mutation and identify specific new therapeutic targets.