A culture method with berbamine, a plant alkaloid, enhances CAR-T cell efficacy through modulating cellular metabolism.

Memory T cells demonstrate superior in vivo persistence and antitumor efficacy. However, methods for manufacturing less differentiated T cells are not yet well-established. Here, we show that producing chimeric antigen receptor (CAR)-T cells using berbamine (BBM), a natural compound found in the Chinese herbal medicine Berberis amurensis, enhances the antitumor efficacy of CAR-T cells. BBM is identified through cell-based screening of chemical compounds using induced pluripotent stem cell-derived T cells, leading to improved viability with a memory T cell phenotype. Transcriptomics and metabolomics using stem cell memory T cells reveal that BBM broadly enhances lipid metabolism. Furthermore, the addition of BBM downregulates the phosphorylation of p38 mitogen-activated protein kinase and enhanced mitochondrial respiration. CD19-CAR-T cells cultured with BBM also extend the survival of leukaemia mouse models due to their superior in vivo persistence. This technology offers a straightforward approach to enhancing the antitumor efficacy of CAR-T cells.

Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting.

With the current interest in cultured meat, mammalian cell-based meat has mostly been unstructured. There is thus still a high demand for artificial steak-like meat. We demonstrate in vitro construction of engineered steak-like tissue assembled of three types of bovine cell fibers (muscle, fat, and vessel). Because actual meat is an aligned assembly of the fibers connected to the tendon for the actions of contraction and relaxation, tendon-gel integrated bioprinting was developed to construct tendon-like gels. In this study, a total of 72 fibers comprising 42 muscles, 28 adipose tissues, and 2 blood capillaries were constructed by tendon-gel integrated bioprinting and manually assembled to fabricate steak-like meat with a diameter of 5 mm and a length of 10 mm inspired by a meat cut. The developed tendon-gel integrated bioprinting here could be a promising technology for the fabrication of the desired types of steak-like cultured meats.

The soluble form of BMPRIB is a novel therapeutic candidate for treating bone related disorders.

Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the TGF-beta superfamily. Recently, several soluble BMP receptors, such as ActRIIA-Fc, ActRIIB-Fc, and ALK1-Fc, are undergoing clinical trials. Both BMPRIA and BMPRIB are type I BMP receptors, and while BMPRIA-Fc has been reported to have bone-increasing properties, there have been no investigations concerning the biological functions of BMPRIB-Fc. Therefore, comparing the effects of BMPRIA-Fc and BMPRIB-Fc in vivo should be helpful in revealing the differences in biological function between BMPRIA and BMPRIB, and would also aid in the evaluation of BMPRIB-Fc as a therapeutic agent. Here, we produced Tg chimeras in which BMPRIA-Fc and BMPRIB-Fc proteins circulated at high concentrations (36.8-121.4 µg/mL). Both Tg chimeras showed a significant increase of bone volume and strength. Using histological analysis, adenoma of the glandular stomach was observed only in BMPRIA-Fc chimeras suggesting the tumorigenic activity of this protein. Administration of recombinant BMPRIB-Fc protein to normal mice also increased bone volumes. Finally, treatment with BMPRIB-Fc decreased the area of osteolytic regions in a mouse model of breast cancer metastasis. In conclusion, our data suggest that BMPRIB-Fc can be used for the treatment of bone-related disorders with a lower risk than BMPRIA-Fc.

Identification of osteoblast stimulating factor 5 as a negative regulator in the B-lymphopoietic niche.

Recent studies have revealed the crucial role of the niche which supports B-lymphocyte differentiation from hematopoietic stem cells. In this study, we aimed to identify a novel regulator of B lymphopoiesis secreted in the specific niche using the signal sequence trap method. Among the identified proteins from MS5 stromal cells, expression of pleiotrophin, placental proliferin 2, and osteoblast stimulating factor 5 (OSF-5) was dominantly high in several stromal cell lines. We found that OSF-5 suppressed early B lymphopoiesis in transgenic mice producing the target protein. The number of pre-B and immature B cells was reduced by more than half compared with control in the transgenic mice. In vitro studies showed that a secreted variant of OSF-5 inhibited the proliferation and colony formation of pre-B cells, whereas cell-intrinsic form had no influence on B lymphopoiesis. The main components of the B-lymphopoietic niche, osteoblasts in mice and mesenchymal cells in humans, are primary producers of OSF-5. These results define a novel mechanism of B lymphopoiesis in bone marrow. In the specific niche, B-lymphocyte differentiation is fine-tuned by negative regulators as well as supportive factors.

Estrogen-inducible sFRP5 inhibits early B-lymphopoiesis in vivo, but not during pregnancy.

Mammals have evolved to protect their offspring during early fetal development. Elaborated mechanisms induce tolerance in the maternal immune system for the fetus. Female hormones, mainly estrogen, play a role in suppressing maternal lymphopoiesis. However, the molecular mechanisms involved in the maternal immune tolerance are largely unknown. Here, we show that estrogen-induced soluble Frizzled-related proteins (sFRPs), and particularly sFRP5, suppress B-lymphopoiesis in vivo in transgenic mice. Mice overexpressing sFRP5 had fewer B-lymphocytes in the peripheral blood and spleen. High levels of sFRP5 inhibited early B-cell differentiation in the bone marrow (BM), resulting in the accumulation of cells with a common lymphoid progenitor (CLP) phenotype. Conversely, sFRP5 deficiency reduced the number of hematopoietic stem cells (HSCs) and primitive lymphoid progenitors in the BM, particularly when estrogen was administered. Furthermore, a significant reduction in CLPs and B-lineage-committed progenitors was observed in the BM of sfrp5-null pregnant females. We concluded that, although high sFRP5 expression inhibits B-lymphopoiesis in vivo, physiologically, it contributes to the preservation of very primitive lymphopoietic progenitors, including HSCs, under high estrogen levels. Thus, sFRP5 regulates early lympho-hematopoiesis in the maternal BM, but the maternal-fetal immune tolerance still involves other molecular mechanisms that remain to be uncovered.

Adult-specific systemic over-expression reveals novel in vivo effects of the soluble forms of ActRIIA, ActRIIB and BMPRII.

Bone morphogenetic proteins (BMPs)/growth differentiation factors (GDFs), which belong to the TGF-beta superfamily, are pleiotropic factors that play a role in regulating the embryonic development and postnatal homeostasis of various organs and tissues by controlling cellular differentiation, proliferation and apoptosis. Conventional transgenic and knockout (KO) mouse approaches have provided only limited information regarding the in vivo functions of BMP signaling in adult animals due to the effects on prenatal development and the difficulty in manipulating multiligand signals simultaneously. We recently produced transgenic chimeric mice(Tg chimeras) in which the soluble IgG1-Fc fusion protein of three BMP type II receptors (ActRIIA, ActRIIB, BMPRII) was highly circulated (281-709 µg/ml), specifically in adult mouse blood. Since each BMP receptor can bind to multiple BMP ligands, these Tg chimeras should be useful to investigate the effects of trapping multiple BMP ligands. Remarkably, some phenotypes were unexpected based on previous studies, such as KO mouse analyses, presumably representing the effects of the multiple ligand trapping. These phenotypes included increased red blood cells (RBCs) and decreased viability in adults. In a further study, we focused on the phenotype of increased RBCs and found that extramedullary hematopoiesis in the spleen, not in the bone marrow, was increased using histological and flow cytometric analyses. Although it remains to be elucidated whether the transgene products affect the tissues directly or indirectly, our data provide novel and important insight into the biological functions of the soluble IgG1-Fc fusion protein of three BMP type II receptors in adults, and our approach should have broad applications to research on other ligand receptor families and studies involving mouse models.

Targeted deletion of the tybe IIb Na(+)-dependent Pi-co-transporter, NaPi-IIb, results in early embryonic lethality.

NaPi-IIb encodes a Na(+)-dependent Pi co-transporter, which is expressed in various adult tissues and mediates transport of extracellular Pi ions coupling with Na(+) ion. To define the role of NaPi-IIbin vivo, NaPi-IIb gene deficient mice were generated utilizing targeted mutagenesis, yielding viable, heterozygous NaPi-IIb mice. In contrast, homozygous NaPi-IIb mice died in utero soon after implantation, indicating that NaPi-IIb was essential for early embryonic development. In situ hybridization revealed NaPi-IIb mRNA expression in the parietal endoderm, followed by the visceral endoderm, at a time point prior to establishment of a functioning chorio-allantoic placenta. At the time point of functional placenta development, the main site of NaPi-IIb production resided in the labyrinthine zone, where embryonic and maternal circulations were in closest contact. Expression patterns of NaPi-IIb suggest that NaPi-IIb plays an important role in Pi absorption from maternal circulation.

Craniofacial malformation in R-spondin2 knockout mice.

In vertebrates, craniofacial formation is accomplished by synergistic interaction of many small elements which are generated independently from distinct germ layers. Because of its complexity, the imbalance of one signaling cascade such as Wnt/beta-catenin pathway easily leads to craniofacial malformation, which is the most frequent birth defect in humans. To investigate the developmental role of a newly identified activator of Wnt/beta-catenin signaling, Rspo2, we generated and characterized Rspo2(-/-) mice. We found CLP with mild facial skeletal defects in Rspo2(-/-) mice. Additionally, Rspo2(-/-) mice also exhibited distal limb loss and lung hypoplasia, and died immediately after birth with respiratory failure. We showed the apparent reduction of Wnt/beta-catenin signaling activity at the branchial arch and the apical ectodermal ridge in Rspo2(-/-) mice. These findings indicate that Rspo2 regulates midfacial, limb, and lung morphogenesis during development through the Wnt/beta-catenin signaling.

R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling.

In mammals, female development has traditionally been considered a default process in the absence of the testis-determining gene, Sry. Recently, it has been documented that the gene for R-spondin1 (RSPO1), a novel class of soluble activator for Wnt/beta-catenin signaling, is mutated in two Italian families with female-to-male (XX) sex reversal. To elucidate the role of Rspo1 as a candidate female-determining gene in a mouse model, we generated Rspo1-null (Rspo1(-/-)) mice and found that Rspo1(-/-) XX mice displayed masculinized features including pseudohermaphroditism in genital ducts, depletion of fetal oocytes, male-specific coelomic vessel formation and ectopic testosterone production in the ovaries. Thus, although Rspo1 is required to fully suppress the male differentiation program and to maintain germ cell survival during the development of XX gonads, the loss of its activity has proved to be insufficient to cause complete XX sex reversal in mice. Interestingly, these partial sex-reversed phenotypes of Rspo1(-/-) XX mice recapitulated those of previously described Wnt-4(-/-) XX mice. In accordance with this finding, the expression of Wnt-4 and its downstream genes was deregulated in early Rspo1(-/-) XX gonads, suggesting that Rspo1 may participate in suppressing the male pathway in the absence of Sry and maintaining oocyte survival through positively regulating Wnt-4 signaling.

R-Spondin proteins: a novel link to beta-catenin activation.

The R-spondin (Rspo) protein family is a recently described group of four distinct human secreted proteins. Reported activities for Rspo proteins include essential roles in vertebrate development and their ligand-type activities overlap substantially with those of the canonical Wnt ligands in that both Rspo and canonical Wnt signaling result in the activation of beta-catenin. In a general functional screen for human secreted proteins using transgenic mouse models, we identified human R-spondin1 (hRspo1) protein as a potent and specific mitogen for the gastrointestinal epithelium and demonstrated a potential therapeutic application for the protein in mouse models of cancer therapy-induced mucositis. In contrast to previous studies, our data indicated only partial overlap between Wnt and Rspo ligand activities, suggesting that there may be independent receptor/signaling pathways for Rspo proteins that intersect those of Wnt at the level of beta-catenin. Here we summarize the current reported data on the Rspo family and discuss these results in terms of alternate mechanisms of action. We have extended our observations on the potential therapeutic application of Rspo proteins by showing that all four human Rspo family members are capable of inducing epithelial proliferation and report the first non-vertebrate Rspo family member.

Mitogenic influence of human R-spondin1 on the intestinal epithelium.

Several described growth factors influence the proliferation and regeneration of the intestinal epithelium. Using a transgenic mouse model, we identified a human gene, R-spondin1, with potent and specific proliferative effects on intestinal crypt cells. Human R-spondin1 (hRSpo1) is a thrombospondin domain-containing protein expressed in enteroendocrine cells as well as in epithelial cells in various tissues. Upon injection into mice, the protein induced rapid onset of crypt cell proliferation involving beta-catenin stabilization, possibly by a process that is distinct from the canonical Wnt-mediated signaling pathway. The protein also displayed efficacy in a model of chemotherapy-induced intestinal mucositis and may have therapeutic application in gastrointestinal diseases.

Generation of chromosome-specific monoclonal antibodies using in vitro-differentiated transchromosomic mouse embryonic stem cells.

Monoclonal antibodies (MoAbs) recognizing lineage- and stage-specific human cell-surface antigens are valuable reagents for the characterization and isolation of various specialized cell populations derived from human embryonic stem cells (hESCs). In this report, we examined the use of in vitro differentiated transchromosomic mouse embryonic stem cells (TC-ESCs) as immunogens to obtain MoAbs against human cell-surface antigens. Immunization of a neural-cell population derived from differentiating human chromosome 4 and 11 TC-ESCs resulted in two chromosome-specific MoAbs, h4-neural1 and h11-neural1, respectively. The staining profiles of differentiated TC-ESCs and human embryonic carcinoma cells with these MoAbs were similar to the expression profile of nestin, a well-characterized intracellular marker for neural progenitor cells. We also described the successful purification and identification of the gene for h4-neural1 antigen (CD133, 4p15.32) with immunoaffinity chromatography. This procedure may have significant utility in generating MoAbs useful for understanding the mechanism that regulates the in vitro differentiation of hESCs.

Vitamin D receptor-independent FGF23 actions in regulating phosphate and vitamin D metabolism.

FGF23 suppresses both serum phosphate and 1,25-dihydroxyvitamin D [1,25D] levels in vivo. Because 1,25D itself is a potent regulator of phosphate metabolism, it has remained unclear whether FGF23-induced changes in phosphate metabolism were caused by a 1,25D-independent mechanism. To address this issue, we intravenously administered recombinant FGF23 to vitamin D receptor (VDR) null (KO) mice as a rapid bolus injection and evaluated the early effects of FGF23. Administration of recombinant FGF23 further decreased the serum phosphate level in VDR KO mice, accompanied by a reduction in renal sodium-phosphate cotransporter type IIa (NaPi2a) protein abundance and a reduced renal 25-hydroxyvitamin D-1alpha-hydroxylase (1alphaOHase) mRNA level. Thus FGF23-induced changes in NaPi2a and 1alphaOHase expression are independent of the 1,25D/VDR system. However, 24-hydroxylase (24OHase) mRNA expression remained undetectable by the treatment with FGF23. We also analyzed the regulatory mechanism for FGF23 expression. The serum FGF23 level was almost undetectable in VDR KO mice, whereas dietary calcium supplementation significantly increased circulatory levels of FGF23 and its mRNA abundance in bone. This finding indicates that calcium is another determinant of FGF23 production that occurs independently of the VDR-mediated mechanism. In contrast, dietary phosphate supplementation failed to induce FGF23 expression in the absence of VDR, whereas marked elevation in circulatory FGF23 was observed in wild-type mice fed with a high-phosphate diet. Taken together, FGF23 works, at least in part, in a VDR-independent manner, and FGF23 production is also regulated by multiple mechanisms involving VDR-independent pathways.

A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins.

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) kappa locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igkappa region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras.

A new model mouse for Duchenne muscular dystrophy produced by 2.4 Mb deletion of dystrophin gene using Cre-loxP recombination system.

Duchenne muscular dystrophy (DMD) is caused by mutation in the 2.4-Mb dystrophin (DMD) gene . This gene encodes a number of tissue-specific isoforms of dystrophin generated by transcription from at least seven promoters and also by alternative splicing. We deleted entire genomic region of the DMD gene on mouse chromosome X using a Cre-loxP recombination system. Introduction of a loxP site in dystrophin's first and last exon by homologous recombination in mouse embryonic stem (ES) cells generated "DMD-floxed" (flanked by loxP sites) ES cells, which we subjected to Cre-mediated excision leading to establishment of "DMD-null" ES cell lines. The DMD-null mice produced from the DMD-null ES cells were viable but displayed severe muscular hypertrophy and dystrophy. In addition to the muscular impairment, the DMD-null mouse exhibited some behavioral abnormality and male sterility. The DMD-floxed mice produced from the DMD-floxed ES cells were viable, phenotypically normal, and were born with the expected Mendelian frequency, despite the absence of brain (cortical)-type dystrophin (Dp427c) expression. Since production of multiple dystrophin isoforms due to alternative splicing or exon skipping is totally prevented in the DMD-null mouse, these new mutants will provide an improved model system for functional studies of dystrophin and its isoforms.

Sequential targeting of the genes encoding immunoglobulin-mu and prion protein in cattle.

Gene targeting is accomplished using embryonic stem cells in the mouse but has been successful, only using primary somatic cells followed by embryonic cloning, in other species. Gene targeting in somatic cells versus embryonic stem cells is a challenge; consequently, there are few reported successes and none include the targeting of transcriptionally silent genes or double targeting to produce homozygotes. Here, we report a sequential gene targeting system for primary fibroblast cells that we used to knock out both alleles of a silent gene, the bovine gene encoding immunoglobulin-mu (IGHM), and produce both heterozygous and homozygous knockout calves. We also carried out sequential knockout targeting of both alleles of a gene that is active in fibroblasts, encoding the bovine prion protein (PRNP), in the same genetic line to produce doubly homozygous knockout fetuses. The sequential gene targeting system we used alleviates the need for germline transmission for complex genetic modifications and should be broadly applicable to gene functional analysis and to biomedical and agricultural applications.

Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism.

Inorganic phosphate is essential for ECM mineralization and also as a constituent of important molecules in cellular metabolism. Investigations of several hypophosphatemic diseases indicated that a hormone-like molecule probably regulates serum phosphate concentration. FGF23 has recently been recognized as playing important pathophysiological roles in several hypophosphatemic diseases. We present here the evidence that FGF23 is a physiological regulator of serum phosphate and 1,25-dihydroxyvitamin D (1,25[OH]2D) by generating FGF23-null mice. Disruption of the Fgf23 gene did not result in embryonic lethality, although homozygous mice showed severe growth retardation with abnormal bone phenotype and markedly short life span. The Fgf23(-/-) mice displayed significantly high serum phosphate with increased renal phosphate reabsorption. They also showed an elevation in serum 1,25(OH)2D that was due to the enhanced expression of renal 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase) from 10 days of age. These phenotypes could not be explained by currently known regulators of mineral homeostasis, indicating that FGF23 is essential for normal phosphate and vitamin D metabolism.