Hypoimmunogenic human iPSCs expressing HLA-G, PD-L1, and PD-L2 evade innate and adaptive immunity.

BACKGROUND: The human induced pluripotent stem cells (hiPSCs) can generate all the cells composing the human body, theoretically. Therefore, hiPSCs are thought to be a candidate source of stem cells for regenerative medicine. The major challenge of allogeneic hiPSC-derived cell products is their immunogenicity. The hypoimmunogenic cell strategy is allogenic cell therapy without using immune suppressants. Advances in gene engineering technology now permit the generation of hypoimmunogenic cells to avoid allogeneic immune rejection. In this study, we generated a hypoimmunogenic hiPSC (HyPSC) clone that had diminished expression of human leukocyte antigen (HLA) class Ia and class II and expressed immune checkpoint molecules and a safety switch. METHODS: First, we generated HLA class Ia and class II double knockout (HLA class Ia/II DKO) hiPSCs. Then, a HyPSC clone was generated by introducing exogenous ß-2-microglobulin (B2M), HLA-G, PD-L1, and PD-L2 genes, and the Rapamycin-activated Caspase 9 (RapaCasp9)-based suicide gene as a safety switch into the HLA class Ia/II DKO hiPSCs. The characteristics and immunogenicity of the HyPSCs and their derivatives were analyzed. RESULTS: We found that the expression of HLA-G on the cell surface can be enhanced by introducing the exogenous HLA-G gene along with B2M gene into HLA class Ia/II DKO hiPSCs. The HyPSCs retained a normal karyotype and had the characteristics of pluripotent stem cells. Moreover, the HyPSCs could differentiate into cells of all three germ layer lineages including CD45+ hematopoietic progenitor cells (HPCs), functional endothelial cells, and hepatocytes. The HyPSCs-derived HPCs exhibited the ability to evade innate and adaptive immunity. Further, we demonstrated that RapaCasp9 could be used as a safety switch in vitro and in vivo. CONCLUSION: The HLA class Ia/II DKO hiPSCs armed with HLA-G, PD-L1, PD-L2, and RapaCasp9 molecules are a potential source of stem cells for allogeneic transplantation.

A case of acute cholecystitis with abnormally high CA19-9.

BACKGROUND: CA19-9 is strongly expressed in malignant tumors of the digestive system and is widely used as a marker for gastrointestinal cancer. In this report, we describe a case of acute cholecystitis in which CA19-9 was markedly elevated. CASE PRESENTATION: A 53-year-old man was admitted to our hospital with a diagnosis of acute cholecystitis after being referred to our hospital with a chief complaint of fever and right hypochondrial pain. CA19-9 was abnormally high at 17,539.1 U/ml. Although the possibility of malignancy was considered, there was no obvious malignant lesion on imaging; the patient was diagnosed with cholecystitis, and laparoscopic cholecystectomy was performed the day after admission. The surgical specimen showed no malignant findings either grossly or in the final pathological examination. There were no complications in the patient's postoperative course, and he was discharged from the hospital on the third postoperative day. CA19-9 level quickly returned to within normal range after surgery. CONCLUSIONS: In acute cholecystitis, CA19-9 levels exceeding 10,000 U/ml are very rare. We report a case of acute cholecystitis without malignant findings despite a high CA19-9 level.

Enhanced survival of hypoimmunogenic otic progenitors following intracochlear xenotransplantation: repercussions for stem cell therapy in hearing loss models.

Stem cell replacement holds the potential for sensorineural hearing loss (SNHL) treatment. However, its translation into clinical practice requires strategies for improving stem cell survival following intracochlear transplantation. Considering recent findings showing that the inner ear contains a resident population of immune cells, we hypothesized that immune evasion would improve the survival and residence time of transplanted stem cells in the cochlea, potentially leading to better outcomes. To test this, we leveraged genetic engineering techniques to develop hypoimmunogenic human-induced pluripotent stem cells (hi-iPSC), which lack human leukocyte antigen expression. We found that gene editing does not affect the biological properties of hi-iPSCs, including their capacity to differentiate into otic neural progenitors (ONPs). Compared to wild-type ONPs, more hypoimmunogenic ONPs (derived from hi-iPSCs) were found in the inner ear of immunocompetent mice ten days following cochlear xenotransplantation. This approach may open a new avenue for experimental and clinical SNHL treatments.

Report of the international conference on manufacturing and testing of pluripotent stem cells.

Sessions included an overview of past cell therapy (CT) conferences sponsored by the International Alliance for Biological Standardization (IABS). The sessions highlighted challenges in the field of human pluripotent stem cells (hPSCs) and also addressed specific points on manufacturing, bioanalytics and comparability, tumorigenicity testing, storage, and shipping. Panel discussions complemented the presentations. The conference concluded that a range of new standardization groups is emerging that could help the field, but ways must be found to ensure that these efforts are coordinated. In addition, there are opportunities for regulatory convergence starting with a gap analysis of existing guidelines to determine what might be missing and what issues might be creating divergence. More specific global regulatory guidance, preferably from WHO, would be welcome. IABS and the California Institute for Regenerative Medicine (CIRM) will explore with stakeholders the development of a practical and innovative road map to support early CT product (CTP) developers.

The mTOR pathway controls cell proliferation by regulating the FoxO3a transcription factor via SGK1 kinase.

The mechanistic target of rapamycin (mTOR) functions as a component of two large complexes, mTORC1 and mTORC2, which play crucial roles in regulating cell growth and homeostasis. However, the molecular mechanisms by which mTOR controls cell proliferation remain elusive. Here we show that the FoxO3a transcription factor is coordinately regulated by mTORC1 and mTORC2, and plays a crucial role in controlling cell proliferation. To dissect mTOR signaling, mTORC1 was specifically inactivated by depleting p18, an essential anchor of mTORC1 on lysosomes. mTORC1 inactivation caused a marked retardation of cell proliferation, which was associated with upregulation of cyclin-dependent kinase inhibitors (CDKIs). Although Akt was activated by mTORC1 inactivation, FoxO3a was upregulated via an epigenetic mechanism and hypophosphorylated at Ser314, which resulted in its nuclear accumulation. Consistently, mTORC1 inactivation induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1), the kinase responsible for Ser314 phosphorylation. Expression of FoxO3a mutated at Ser314 suppressed cell proliferation by inducing CDKI expression. SGK1 overexpression suppressed CDKI expression in p18-deficient cells, whereas SGK1 knockdown induced CDKI expression in wild-type cells, resulting in the suppression of cell proliferation. These results suggest that mTORC1, in coordination with mTORC2, controls cell proliferation by regulating FoxO3a gene expression and SGK1-mediated phosphorylation of FoxO3a at Ser314.

Aprepitant plus granisetron and dexamethasone for prevention of chemotherapy-induced nausea and vomiting in patients with gastric cancer treated with S-1 plus cisplatin.

BACKGROUND: We aimed to evaluate the efficacy of a new combination antiemetic therapy comprising aprepitant, granisetron, and dexamethasone in gastric cancer patients undergoing chemotherapy with cisplatin and S-1. METHODS: Gastric cancer patients scheduled to receive their first course of chemotherapy with cisplatin (60 mg/m(2)) and S-1 (80 mg/m(2)) were treated with a new combination antiemetic therapy aprepitant, granisetron, and dexamethasone on day 1; aprepitant and dexamethasone on days 2 and 3; and dexamethasone on day 4. The patients reported vomiting, nausea, use of rescue therapy, and change in the amount of diet intake, and completed the Functional Living Index-Emesis (FLIE) questionnaire. The primary endpoint was complete response (CR; no emesis and use of no rescue antiemetics) during the overall study phase (0-120 h after cisplatin administration). The secondary endpoints included complete protection (CP; CR plus no significant nausea); change in the amount of diet intake; and the impact of chemotherapy-induced nausea and vomiting (CINV) on daily life during the overall, acute (0-24 h), and delayed (24-120 h) phases. RESULTS: Fifty-three patients were included. CR was achieved in 88.7, 98.1, and 88.7% of patients in the overall, acute, and delayed phases, respectively. The corresponding rates of CP were 67.9, 96.2, and 67.9%. Approximately half of the patients had some degree of anorexia. FLIE results indicated that 79.5% of patients reported "minimal or no impact of CINV on daily life". CONCLUSIONS: Addition of aprepitant to standard antiemetic therapy was effective in gastric cancer patients undergoing treatment with cisplatin and S-1.

[A case of 5-year survival with combination chemotherapy for advanced colonic cancer with multiple lung and liver metastases and peritoneal dissemination].

A 74-year-old man was admitted to the hospital because of abdominal fullness and constipation. Endoscopy and abdominal computed tomography (CT) scans showed an advanced sigmoid colonic cancer with multiple lung and liver metastases and peritoneal dissemination. Sigmoid resection was performed to relieve ileus. Postoperative chemotherapy was administered for metastatic lesions. We administered systemic chemotherapy with tegafur-uraci(l UFT)/UZEL( 10 months), oxaliplatin with fluorouracil (5-FU) and folinic acid (FOLFOX; 11 courses), irinotecan with 5-FU and folinic acid (FOLFIRI; 13 courses), irinotecan plus S-1 (IRIS; 23 courses), bevacizumab (32 courses), cetuximab (29 courses), and peritoneal infusion of paclitaxel( 65 courses). The patient survived for 5 years after the initial treatment.

An anilinoquinazoline derivative inhibits tumor growth through interaction with hCAP-G2, a subunit of condensin II.

We screened 46 novel anilinoquinazoline derivatives for activity to inhibit proliferation of a panel of human cancer cell lines. Among them, Q15 showed potent in vitro growth-inhibitory activity towards cancer cell lines derived from colorectal cancer, lung cancer and multiple myeloma. It also showed antitumor activity towards multiple myeloma KMS34 tumor xenografts in lcr/scid mice in vivo. Unlike the known anilinoquinazoline derivative gefitinib, Q15 did not inhibit cytokine-mediated intracellular tyrosine phosphorylation. Using our mRNA display technology, we identified hCAP-G2, a subunit of condensin II complex, which is regarded as a key player in mitotic chromosome condensation, as a Q15 binding partner. Immunofluorescence study indicated that Q15 compromises normal segregation of chromosomes, and therefore might induce apoptosis. Thus, our results indicate that hCAP-G2 is a novel therapeutic target for development of drugs active against currently intractable neoplasms.

Down-regulation of the tumor suppressor C-terminal Src kinase (Csk)-binding protein (Cbp)/PAG1 is mediated by epigenetic histone modifications via the mitogen-activated protein kinase (MAPK)/phosphatidylinositol 3-kinase (PI3K) pathway.

The transmembrane adaptor protein Cbp (or PAG1) functions as a suppressor of Src-mediated tumor progression by promoting the inactivation of Src. The expression of Cbp is down-regulated in Src-transformed cells and in various human cancer cells, suggesting a potential role for Cbp as a tumor suppressor. However, the mechanisms underlying the down-regulation of Cbp remain unknown. The present study shows that Cbp expression is down-regulated by epigenetic histone modifications via the MAPK/PI3K pathway. In mouse embryonic fibroblasts, transformation by oncogenic Src and Ras induced a marked down-regulation of Cbp expression. The levels of Cbp expression were inversely correlated with the activity of MEK and Akt, and Cbp down-regulation was suppressed by inhibiting MEK and PI3K. Src transformation did not affect the stability of Cbp mRNA, the transcriptional activity of the cbp promoter, or the DNA methylation status of the cbp promoter CpG islands. However, Cbp expression was restored by treatment with histone deacetylase (HDAC) inhibitors and by siRNA-mediated knockdown of HDAC1/2. Src transformation significantly decreased the acetylation levels of histone H4 and increased the trimethylation levels of histone H3 lysine 27 in the cbp promoter. EGF-induced Cbp down-regulation was also suppressed by inhibiting MEK and HDAC. Furthermore, the inhibition of MEK or HDAC restored Cbp expression in human cancer cells harboring Cbp down-regulation through promoter hypomethylation. These findings suggest that Cbp down-regulation is primarily mediated by epigenetic histone modifications via oncogenic MAPK/PI3K pathways in a subset of cancer cells.

[A case of effective treatment with UFT/LV chemotherapy for para-aortic lymph node metastasis from colonic cancer 8 years after colectomy].

A 77-year-old woman was admitted to our hospital because of para-aortic lymph node swelling pointed out by abdominal CT scan. She had a previous history of colectomy with a diagnosis of ascending colonic cancer 8 years later. Pathological examination was a moderately-differentiated adenocarcinoma with lymph node metastasis (Stage IIIa). PET scan demonstrated hot spots of para-aortic and left supra-clavicular lesions. The serum CEA and CA19-9 levels regained the normal value. No malignancy was recognized by endoscopic examinations of upper and lower gastrointestinal tract. We supported that swelling of lymph nodes were due to lymphoma. Laparotomic biopsy of para-aortic lymph node was done after 6 months. Histologically metastatic adenocarcinoma was recognized. We performed systemic chemotherapy of UFT and LV on recurrent colonic cancer. After 9 courses, spots of para-aortic and left supra-clavicular lesions disappeared on the PET scan. Lymph node metastasis was not found by CT scan 20 months after beginning chemotherapy. Thus, we consider that this therapy was recommendable for the treatment of an older adult patient with recurrent colonic cancer.

Safety and efficacy of bidirectional chemotherapy for treatment of patients with peritoneal dissemination from gastric cancer: Selection for cytoreductive surgery.

There is no standard treatment for peritoneal carcinomatosis (PC) from gastric cancer. New bidirectional chemotherapy (neoadjuvant intraperitoneal-systemic chemotherapy protocol (NIPS)) was developed. The aim of the present study was to assess the safety and efficacy of NIPS and to show the selection for cytoreductive surgery on PC from gastric cancer. Seventy-nine patients with PC from gastric cancer were treated with NIPS. A peritoneal port system was introduced into the abdominal cavity. The peritoneal wash cytological examination through a port was done before and after NIPS. The patients were treated with oral TS-1 twice a daily for 21 days, followed by a 1-week rest. On day 1, 8, and 15 from the start of oral TS-1 administration, 30 mg/m(2) of Docetaxel and 30 mg/m(2) of cisplatinum with 500 ml of saline were introduced into the peritoneal cavity through the port. A median course of oral TS-1 was 2.1 course and a median time of IP chemoterapy was 5.8. Peritoneal free cancer cells (PFCCs) had been detected in 65 (82.2%) patients before NIPS, and the positive cytology changed to be negative in 41 (63.0%) patients after NIPS. After NIPS, 41 patients underwent laparotomy, and complete cytoreduction was done in 32 (78%) patients. Complete cytoreduction was done in 27 (51.9%) of 52 patients with negative cytology but in only 4 (14.8%) of 27 patients with positive cytology (P < 0.001). Patients with negative cytology after NIPS survived significantly longer than those with positive cytology. The adverse effects after NIPS were mild and there was no treatment-related deaths. The grade 3/4 hematological adverse effects were found in 2 (2.6%) patients. Grade 3 renal toxicity and port site infection was found in three patients, respectively. NIPS using a port system is a safe and effective treatment for PC. Peritoneal wash cytology through a port system is a good indicator to select the patients to perform cytoreductive surgery.

[A resected case of effective treatment with S-1/gemcitabine and Paclitaxel combination chemotherapy for advanced pancreatic cancer with peritoneal and liver metastases].

A 58-year-old woman was admitted to the hospital because of abdominal fullness and anorexia. Abdominal CT scan revealed pancreatic cancer with massive ascites showing peritoneal dissemination, combined with lymph node and liver metastases. Cytology of ascites showed malignancy. Abnormally high values of CA19-9 (1,908 U/mL) and CA125 (545 U/mL) were detected in serum. Histologically, metastatic adenocarcinoma was recognized by laparoscopic biopsy of peritoneal dissemination. We performed systemic chemotherapy of S-1, gemcitabine and peritoneal infusion of paclitaxel for nonresected pancreatic cancer. After 5 courses, ascites disappeared and the serum CA19-9 and CA125 levels regained their normal value. Peritoneal seeding was not found by second laparoscopic examination 20 months after beginning chemotherapy. Thus, we consider the patient had an effective response, and performed distal pancreatectomy and proximal resection of the stomach. Histological examination of the primary lesion revealed no cancer cells where fibrosis presented.

[A case of nonresected gastric cancer with peritoneal dissemination maintained on TS-1, cisplatin (CDDP) and docetaxel combination chemotherapy with good QOL].

A 64-year-old woman was admitted to the hospital for abdominal fullness and constipation. In the pelvic cavity, an abdominal CT scan revealed massive ascites showing malignancy on histological examination. Upper GI endoscopy revealed type 3 gastric cancer from the anglus to the cardia. A barium-enema showed a stenotic lesion at the sigmoid colon due to peritoneal dissemination. An abnormally high CA125(1,400 mg/ml) level was detected in serum. We performed systemic chemotherapy of TS-1, CDDP and peritoneal infusion of docetaxel on the nonresected gastric cancer with peritoneal dissemination. After 2 cycles, cytology of ascites revealed no malignancy, and the serum CA125 value regained its normal level. After 3 cycles, the killer cell effect was recognized by laparoscopic examination and the stenotic change of sigmoid colon had almost disappeared. The patient clinically achieved good QOL by this method, which was very effective for nonresected gastric cancer with peritoneal dissemination.

Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression.

The pluripotential cell-specific gene Nanog encodes a homeodomain-bearing transcription factor required for maintaining the undifferentiated state of stem cells. However, the molecular mechanisms that regulate Nanog gene expression are largely unknown. To address this important issue, we used luciferase assays to monitor the relative activities of deletion fragments from the 5'-flanking region of the gene. An adjacent pair of highly conserved Octamer- and Sox-binding sites was found to be essential for activating pluripotential state-specific gene expression. Furthermore, the 5'-end fragment encompassing the Octamer/Sox element was sufficient for inducing the proper expression of a green fluorescent protein reporter gene even in human embryonic stem (ES) cells. The potential of OCT4 and SOX2 to bind to this element was verified by electrophoretic mobility shift assays with extracts from F9 embryonal carcinoma cells and embryonic germ cells derived from embryonic day 12.5 embryos. However, in ES cell extracts, a complex of OCT4 with an undefined factor preferentially bound to the Octamer/Sox element. Thus, Nanog transcription may be regulated through an interaction between Oct4 and Sox2 or a novel pluripotential cell-specific Sox element-binding factor which is prominent in ES cells.

Pluripotential competence of cells associated with Nanog activity.

Nanog is a novel pluripotential cell-specific gene that plays a crucial role in maintaining the undifferentiated state of early postimplantation embryos and embryonic stem (ES) cells. We have explored the expression pattern and function of Nanog and a Nanog-homologue, Nanog-ps1.Nanog-ps1 was mapped on Chromosome 7 and shown to be a pseudogene. Immunocytochemical analysis in vivo showed that the NANOG protein was absent in unfertilized oocytes, and was detected in cells of morula-stage embryos, the inner cell mass of blastocysts and the epiblast of E6.5 and E7.5 embryos, but not in primordial germ cells of early postimplantation embryos. In monkey and human ES cells, NANOG expression was restricted to undifferentiated cells. Furthermore, reactivation of the somatic cell-derived Nanog was tightly linked with nuclear reprogramming induced by cell hybridization with ES cells and by nuclear transplantation into enucleated oocytes. Notably, mouse Nanog (+/-) ES cells, which produced approximately half the amount of NANOG produced by wild-type ES cells, readily differentiated to multi-lineage cells in culture medium including LIF. The labile undifferentiated state was fully rescued by constitutive expression of exogenous Nanog. Thus, the activity of Nanog is tightly correlated with an undifferentiated state of cells even in nuclear reprogrammed somatic cells. Nanog may function as a key regulator for sustaining pluripotency in a dose-dependent manner.

Feasibility study of adjuvant chemotherapy with S-1 (TS-1; tegafur, gimeracil, oteracil potassium) for gastric cancer.

BACKGROUND: We conducted a feasibility study using S-1, a novel oral derivative of 5-fluorouracil, as postoperative adjuvant chemotherapy for curatively resected gastric cancer patients. METHODS: Adjuvant chemotherapy consisted of eight courses (4-week administration and 2-week withdrawal) of S-1, at 80-120 mg/body per day. Forty-one patients from 11 institutions were enrolled in this pilot study, from November 1999 to October 2000. RESULTS: Thirty-five patients were eligible. In 7 patients, S-1 administration was discontinued due to recurrence. Among the 28 patients without recurrence, the planned eight courses of S-1 were administered to 17 patients (60.7%). In 4 patients, S-1 administration was discontinued due to subjective symptoms, such as anorexia, in the first course. Adverse reactions such as neutropenia, leukopenia, elevated total bilirubin, anorexia, general fatigue, diarrhea, nausea, and stomatitis were seen in more than half of the patients. Although grade 3 neutropenia (29.3%), leukopenia (9.8%), and diarrhea (9.8%) were observed, no grade 4 adverse effects appeared. Compared with the treatment of unresectable or recurrent gastric cancer with S-1, the incidence of adverse reactions in the adjuvant setting was slightly higher, probably due to the influence of gastrectomy. CONCLUSION: Except for the early development of anorexia, most likely due to adverse effects of surgery, postoperative administration of S-1 for 1 year seems feasible as adjuvant chemotherapy for gastric cancer.

Pedunculated giant gastrointestinal stromal tumor of the stomach showing extragastric growth: report of a case.

We report an unusual case of a pedunculated gastrointestinal stromal tumor (GIST) of the stomach with extragastric growth. An 84-year-old woman was referred to our hospital for investigation of anemia. Barium meal examination showed a defect occupying the lesser curvature of the stomach from the middle of the corpus to the fornix and an abdominal computed tomography scan, showed a lesion, 20 cm in diameter, touching the surface of the spleen. Laparotomy revealed a tumor located between the liver and spleen. As the tumor appeared to be connected with the posterior wall of the gastric corpus by a pedicle, it was resected with a pedunculated lesion of the stomach. Based on the pathological findings, the tumor was diagnosed as a GIST of uncommitted type, with low-grade malignancy.

Successful resection of locally advanced rectal carcinoma combined with preoperative chemoradiation.

Chemoradiation increases the local control and colostomy-free survival in patients with advanced anal canal carcinomas. We recently experienced a 50-year-old female patient with locally advanced carcinoma of the rectum invading to the sacrum, which could not be surgically resected. Therefore, we performed a colostomy and transarterial infusion of anti-tumor drugs including 50 mg cisplatin and 10 mg mitomycin C via the inferior mesentery artery in September 1999. She then underwent radiotherapy with a total dose of 64 Gy for the primary tumor. After the radiotherapy the patient received 125 mg/body of 1-leucovorin by 2-hour infusion and, one hour after starting the 1-leucovorin infusion, she received an intravenous bolus of 250 mg/body of 5-fluorouracil. This regimen was conducted biweekly for 13 courses followed by oral administration of 400 mg 5'-deoxyfluorouridine for one year. Since the chemoradiation resulted in a successful response, the patient underwent curative resection of the primary tumor in June 2001. The resected tumor (32 x 35 mm) had clean margins. Although the carcinoma cells had disappeared, fibrous lesions were observed over a broad area. Based on these pathologic findings, the treatment effect was judged as grade Ib.