Induction of functional xeno-free MSCs from human iPSCs via a neural crest cell lineage

iPS細胞から間葉系幹細胞の誘導方法を確立 --動物由来成分を含まず再生医療への利用に期待. 京都大学プレスリリース. 2022-09-15.A new method for inducing mesenchymal stem cells from iPS cells without using animal-derived components. 京都大学プレスリリース. 2022-09-27.Mesenchymal stem/stromal cells (MSCs) are adult multipotent stem cells. Here, we induced MSCs from human induced pluripotent stem cells (iPSCs) via a neural crest cell (NCC) lineage under xeno-free conditions and evaluated their in vivo functions. We modified a previous MSC induction method to work under xeno-free conditions. Bovine serum albumin-containing NCC induction medium and fetal bovine serum-containing MSC induction medium were replaced with xeno-free medium. Through our optimized method, iPSCs differentiated into MSCs with high efficiency. To evaluate their in vivo activities, we transplanted the xeno-free-induced MSCs (XF-iMSCs) into mouse models for bone and skeletal muscle regeneration and confirmed their regenerative potency. These XF-iMSCs mainly promoted the regeneration of surrounding host cells, suggesting that they secrete soluble factors into affected regions. We also found that the peroxidasin and IGF2 secreted by the XF-iMSCs partially contributed to myotube differentiation. These results suggest that XF-iMSCs are important for future applications in regenerative medicine

Synchronous bilateral pheochromocytomas and paraganglioma with novel germline mutation in MAX: a case report

BackgroundRecent advance of genetic testing has contributed to the diagnosis of hereditary pheochromocytoma and paraganglioma (PPGL). The clinical characteristics of hereditary PPGL are varying among the types of mutational genes. It is still difficult to specify the pathognomonic symptoms in the case of rare genetic mutations. Here, we report the case of synchronous bilateral pheochromocytomas and paraganglioma with novel MYC associated factor X (MAX) gene mutation.Case presentationA 24-year-old female had hyperhidrosis and hypertension. Her urine test showed high normetanephrine and vanillylmandelic acid. Enhanced computed tomography revealed three enhanced masses in right adrenal gland, left adrenal gland, and left renal hilus. She was diagnosed with PPGL. Because 123I-metaiodobenzylguanidine scintigraphy indicated the accumulations in the left adrenal gland mass and the left renal hilus mass and not in the right adrenal gland mass, we performed laparoscopic left adrenalectomy and extirpation of the left renal hilus mass to preserve the right adrenocortical function. However, her symptoms recurred shortly after the operation presumably due to unveiling of the activity of the right pheochromocytoma. Following right adrenalectomy as the second operation, the catecholamine levels declined to normal range. Her genetic testing indicated the novel germline mutation in MAX gene (c.70_73 del AAAC/p.Lys24fs*40).ConclusionsMAX germline mutation is recently identified as a rare cause of hereditary PPGL. The deletion mutation in MAX gene in this patient has never reported before. In the case of bilateral pheochromocytomas, the surgical indication should be decided considering each patient’s genetic background. Due to the possibility for other types of malignant tumors, close follow-up is essential for MAX mutation carriers

Development of a novel automatic ascites filtration and concentration equipment with multi‐ring‐type roller pump units for cell‐free and concentrated ascites reinfusion therapy

Cell‐free and concentrated ascites reinfusion therapy (CART) is an effective therapy for refractory ascites. However, CART is difficult to perform as ascites filtration and concentration is a complicated procedure. Moreover, the procedure requires the constant assistance of a clinical engineer or/and the use of an expensive equipment for the multi‐purpose blood processing. Therefore, we developed a CART specialized equipment (mobility CART [M‐CART]) that could be used safely with various safety measures and automatic functions such as automatic washing of clogged filtration filter and self‐regulation of the concentration ratio. Downsizing, lightning of the weight, and automatic processing in M‐CART required the use of newly developed multi‐ring‐type roller pump units. This equipment was approved under Japanese regulations in 2018. In performing 41 sessions of CART (for malignant ascites, 22 sessions; and hepatic ascites, 19 sessions) using this equipment in 17 patients, no serious adverse event occurred. An average of 4494 g of ascites was collected and the total amount of ascites was processed in all the sessions without any trouble. The mean weight of the processed ascites was 560 g and the mean concentration ratio was 8.0. The ascites were processed at a flow rate of 50 mL/min. The mean ascites processing time was 112.5 minutes and a 106.5‐minutes (95.2%) ascites processing was performed automatically. The operator responded to alarms or support information 3.2 times on average (3.1 minutes, 2.1% of ascites processing time). Human errors related to ascites processing were detected by M‐CART at 0.4 times per session on average and were appropriately addressed by the operator. The frequencies of automatic washing of clogged filtration filter and self‐regulation of the concentration ratio were 31.7% and 53.7%, respectively. The mean recovery rates (recovery dose) of protein, albumin, and immunoglobulin G were 72.9%, 72.9%, and 71.2% (65.9 g, 34.9 g, and 13.2 g), respectively. Steroids were administered in 92.7% of the sessions to prevent fever and the mean increase in body temperature was 0.53°C. M‐CART is a compact and lightweight automatic CART specialized equipment that can safely and easily process a large quantity of ascites without the constant assistance of an operator

GFAP-immunoreactive astrocytes in the ventral anterior-lateral complex of the thalamus (VAL) of WT, homozygous <i>Slc19a3</i> KO, and <i>Slc19a3</i> KI mice.

<p>A, C, E, G. Low-magnification images of reactive astrocytes in the thalamus of a WT mouse fed with a conventional diet at postnatal day 35 (A); a homozygous KO mouse fed with thiamine 0.60 mg/100 g food for 5 days (C) and for 12 days (E); and a KI mouse fed with thiamine 0.60 mg/100 g for 14 days (G). B, D, F, and H are high-magnification images of the VAL area (insets) shown in panels of A, C, E, and G, respectively. Scale bars, 500 μm (upper panel) and 100 μm (lower panel).</p

Temporal profile of NeuN-immunostaining in the hippocampus of WT, homozygous <i>Slc19a3</i> KO, and <i>Slc19a3</i> KI mice.

<p>NeuN-immunoreactive neurons in the submedial nucleus of the thalamus (SMT) and ventral anterior-lateral complex of the thalamus (VAL). A. Low magnification of a Nissl-stained brain slice at the level of the hippocampus (HIP). B. High-magnification overview of the specific regions of the thalamic area of a WT mouse. C. Closer view of a region contained in the inset of panel B. D–E. Brain slices of a homozygous KO mouse fed with thiamine 1.71 mg/100 g food for 12 days (D), thiamine 0.6 mg/100 g food for 5 days (E) and for 12 days (F). G–I. Brain slices of a homozygous KI mouse fed with thiamine 1.71 mg/100 g food for 14 days (G), thiamine 0.6 mg/100 g food for 14 days (H) and thiamine 0.27 mg/100 g food for 14 days (I). Scale bar, 200 μm. VPM: ventral posteromedial nucleus of the thalamus, CM: central medial nucleus of the thalamus, VM: ventral medial nucleus of the thalamus, mtt: mammillothalamic tract.</p

Thiamine concentrations in the blood and brain of WT, homozygous <i>Slc19a3</i> KO, and <i>Slc19a3</i> KI mice.

<p>Whole blood and cerebrum homogenates of KO and KI mice were obtained at 5 and 14 days of thiamine restriction, respectively. A. Thiamine concentration in whole blood (nmol/mL). B. Thiamine concentration in cerebrum homogenates (nmol/g wet weight). Bullets, individual thiamine concentrations; and bars, mean values.</p