Targeting mTOR signaling pathways in multiple myeloma: biology and implication for therapy.

Multiple Myeloma (MM), a cancer of terminally differentiated plasma cells, is the second most prevalent hematological malignancy and is incurable due to the inevitable development of drug resistance. Intense protein synthesis is a distinctive trait of MM cells, supporting the massive production of clonal immunoglobulins or free light chains. The mammalian target of rapamycin (mTOR) kinase is appreciated as a master regulator of vital cellular processes, including regulation of metabolism and protein synthesis, and can be found in two multiprotein complexes, mTORC1 and mTORC2. Dysregulation of these complexes is implicated in several types of cancer, including MM. Since mTOR has been shown to be aberrantly activated in a large portion of MM patients and to play a role in stimulating MM cell survival and resistance to several existing therapies, understanding the regulation and functions of the mTOR complexes is vital for the development of more effective therapeutic strategies. This review provides a general overview of the mTOR pathway, discussing key discoveries and recent insights related to the structure and regulation of mTOR complexes. Additionally, we highlight findings on the mechanisms by which mTOR is involved in protein synthesis and delve into mTOR-mediated processes occurring in MM. Finally, we summarize the progress and current challenges of drugs targeting mTOR complexes in MM.

AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity in Acute Myeloid Leukemia.

Rationale: AXL expression has been identified as a prognostic factor in acute myeloid leukemia (AML) and is detectable in approximately 50% of AML patients. In this study, we developed AXL-specific single domain antibodies (sdAbs), cross-reactive for both mouse and human AXL protein, to non-invasively image and treat AXL-expressing cancer cells. Methods: AXL-specific sdAbs were induced by immunizing an alpaca with mouse and human AXL proteins. SdAbs were characterized using ELISA, flow cytometry, surface plasmon resonance and the AlphaFold2 software. A lead compound was selected and labeled with 99mTc for evaluation as a diagnostic tool in mouse models of human (THP-1 cells) or mouse (C1498 cells) AML using SPECT/CT imaging. For therapeutic purposes, the lead compound was fused to a mouse IgG2a-Fc tail and in vitro functionality tests were performed including viability, apoptosis and proliferation assays in human AML cell lines and primary patient samples. Using these in vitro models, its anti-tumor effect was evaluated as a single agent, and in combination with standard of care agents venetoclax or cytarabine. Results: Based on its cell binding potential, cross-reactivity, nanomolar affinity and GAS6/AXL blocking capacity, we selected sdAb20 for further evaluation. Using SPECT/CT imaging, we observed tumor uptake of 99mTc-sdAb20 in mice with AXL-positive THP-1 or C1498 tumors. In THP-1 xenografts, an optimized protocol using pre-injection of cold sdAb20-Fc was required to maximize the tumor-to-background signal. Besides its diagnostic value, we observed a significant reduction in tumor cell proliferation and viability using sdAb20-Fc in vitro. Moreover, combining sdAb20-Fc and cytarabine synergistically induced apoptosis in human AML cell lines, while these effects were less clear when combined with venetoclax. Conclusions: Because of their diagnostic potential, sdAbs could be used to screen patients eligible for AXL-targeted therapy and to follow-up AXL expression during treatment and disease progression. When fused to an Fc-domain, sdAbs acquire additional therapeutic properties that can lead to a multidrug approach for the treatment of AXL-positive cancer patients.

S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma.

Multiple myeloma (MM) is the second most prevalent hematologic malignancy and is incurable because of the inevitable development of drug resistance. Methionine adenosyltransferase 2α (MAT2A) is the primary producer of the methyl donor S-adenosylmethionine (SAM) and several studies have documented MAT2A deregulation in different solid cancers. As the role of MAT2A in MM has not been investigated yet, the aim of this study was to clarify the potential role and underlying molecular mechanisms of MAT2A in MM, exploring new therapeutic options to overcome drug resistance. By analyzing publicly available gene expression profiling data, MAT2A was found to be more highly expressed in patient-derived myeloma cells than in normal bone marrow plasma cells. The expression of MAT2A correlated with an unfavorable prognosis in relapsed patients. MAT2A inhibition in MM cells led to a reduction in intracellular SAM levels, which resulted in impaired cell viability and proliferation, and induction of apoptosis. Further mechanistic investigation demonstrated that MAT2A inhibition inactivated the mTOR-4EBP1 pathway, accompanied by a decrease in protein synthesis. MAT2A targeting in vivo with the small molecule compound FIDAS-5 was able to significantly reduce tumor burden in the 5TGM1 model. Finally, we found that MAT2A inhibition can synergistically enhance the anti-MM effect of the standard-of-care agent bortezomib on both MM cell lines and primary human CD138+ MM cells. In summary, we demonstrate that MAT2A inhibition reduces MM cell proliferation and survival by inhibiting mTOR-mediated protein synthesis. Moreover, our findings suggest that the MAT2A inhibitor FIDAS-5 could be a novel compound to improve bortezomib-based treatment of MM.

Antibiotic resistance characteristics and risk factors analysis of Helicobacter pylori strains isolated from patients in Liaoning Province, an area in North China.

Background: The prevalence of Helicobacter pylori (H. pylori) keeps rising while the eradication rate continues to decline due to the increasing antibiotic resistance. Regional variations of antimicrobial resistance to H. pylori have been recommended by guidelines in recent years. This study aims to investigate the antibiotic resistance rate of H. pylori and its association with infected subjects' characteristics in Liaoning Province, an area in north China. Methods: Gastric tissues from 178 H. pylori positive participants without previous antibiotic use within four weeks were collected for H. pylori culture. Antibiotic susceptibility to furazolidone (AOZ), tetracycline (TC), levofloxacin (LFX), metronidazole (MET), clarithromycin (CLA), and amoxicillin (AMX) were examined with the agar dilution method. Associations between H. pylori resistance and patient characteristics were further analysed. Results: No resistance was observed in AOZ or TC. For LFX, MET, CLA, and AMX, the overall resistance rates were 41.10%, 79.14%, 71.78%, and 22.09% respectively. There were significant differences between resistance to CLA and MALToma (P = 0.021), and between resistance to MET and age (P < 0.001). Conclusions: The primary resistant rates of LEX, MET, CLA, and AMX were relatively high in Liaoning. Treatment effectiveness improvement could be achieved by prior antimicrobial susceptibility tests before antibiotic prescription.

Tuberous Sclerosis Complex With Multiple Organ Tumors: Case Report and Literature Review.

Pancreatic neuroendocrine neoplasms (PNEN) are tumors that originate from neuroendocrine cells. Only about 1% patients are related to mutation of tuberous sclerosis complex gene. Here, we reported a rare case with involvement of multiple organs and space-occupying lesions. Initially, the patient was thought to have metastasis of a pancreatic tumor. However, the patient was diagnosed as pancreatic neuroendocrine tumors, liver perivascular epithelioid tumors, splenic hamartoma, and renal angiomyolipoma by pathological examination after surgery. We performed genetic mutation detection to identify that tuberous sclerosis complex 2 gene presented with a heterozygous variant. Tuberous sclerosis often presents with widespread tumors, but it is less common to present with pancreatic neuroendocrine tumors and liver perivascular tumors as highlighted in the case. So we analyzed the relationship between TSC gene mutations and related tumors. And we also reviewed the current molecular mechanisms and treatments for tuberous sclerosis complex.

System Xc- inhibition blocks bone marrow-multiple myeloma exosomal crosstalk, thereby countering bortezomib resistance.

Multiple myeloma (MM) cells derive proliferative signals from the bone marrow (BM) microenvironment via exosomal crosstalk. Therapeutic strategies targeting this crosstalk are still lacking. Bortezomib resistance in MM cells is linked to elevated expression of xCT (the subunit of system Xc-). Extracellular glutamate released by system Xc- can bind to glutamate metabotropic receptor (GRM) 3, thereby upregulating Rab27-dependent vesicular trafficking. Since Rab27 is also involved in exosome biogenesis, we aimed to investigate the role of system Xc- in exosomal communication between BM stromal cells (BMSCs) and MM cells. We observed that expression of xCT and GRMs was increased after bortezomib treatment in both BMSCs and MM cells. Secretion of glutamate and exosomes was simultaneously enhanced which could be countered by inhibition of system Xc- or GRMs. Moreover, glutamate supplementation increased exosome secretion by increasing expression of Alix, TSG101, Rab27a/b and VAMP7. Importantly, the system Xc- inhibitor sulfasalazine reduced BMSC-induced resistance to bortezomib in MM cells in vitro and enhanced its anti-MM effects in vivo. These findings suggest that system Xc- plays an important role within the BM and could be a potential target in MM.

ER stress arm XBP1s plays a pivotal role in proteasome inhibition-induced bone formation.

BACKGROUND: Bone destruction is a hallmark of multiple myeloma (MM). It has been reported that proteasome inhibitors (PIs) can reduce bone resorption and increase bone formation in MM patients, but the underlying mechanisms remain unclear. METHODS: Mesenchymal stem cells (MSCs) were treated with various doses of PIs, and the effects of bortezomib or carfilzomib on endoplasmic reticulum (ER) stress signaling pathways were analyzed by western blotting and real-time PCR. Alizarin red S (ARS) and alkaline phosphatase (ALP) staining were used to determine the osteogenic differentiation in vitro. Specific inhibitors targeting different ER stress signaling and a Tet-on inducible overexpressing system were used to validate the roles of key ER stress components in regulating osteogenic differentiation of MSCs. Chromatin immunoprecipitation (ChIP) assay was used to evaluate transcription factor-promoter interaction. MicroCT was applied to measure the microarchitecture of bone in model mice in vivo. RESULTS: We found that both PERK-ATF4 and IRE1α-XBP1s ER stress branches are activated during PI-induced osteogenic differentiation. Inhibition of ATF4 or XBP1s signaling can significantly impair PI-induced osteogenic differentiation. Furthermore, we demonstrated that XBP1s can transcriptionally upregulate ATF4 expression and overexpressing XBP1s can induce the expression of ATF4 and other osteogenic differentiation-related genes and therefore drive osteoblast differentiation. MicroCT analysis further demonstrated that inhibition of XBP1s can strikingly abolish bortezomib-induced bone formation in mouse. CONCLUSIONS: These results demonstrated that XBP1s is a master regulator of PI-induced osteoblast differentiation. Activation of IRE1α-XBP1s ER stress signaling can promote osteogenesis, thus providing a novel strategy for the treatment of myeloma bone disease.

Hypoxia Impairs NK Cell Cytotoxicity through SHP-1-Mediated Attenuation of STAT3 and ERK Signaling Pathways.

Natural killer (NK) cells are innate immune effectors with potent antitumor activity. However, tumor cells can create an immunosuppressive microenvironment to escape immune surveillance. Although accumulating evidence indicates that microenvironmental hypoxia plays an important role in favoring tumor development and immune evasion, it remains unclear by what means hypoxia directly impairs NK cell antitumor activity. In this study, we confirmed that hypoxic NK cells showed significantly lower cytotoxicity against tumor cells. Consistent with this finding, we found that the reduction in NK cell cytotoxicity resulting from hypoxia correlated to the lower expression of granzyme B, IFN-γ, and degranulation marker CD107a, as well as activating receptors including NKp30, NKp46, and NKG2D expressed on the surface of NK cells. More importantly, we further demonstrated that a reduction in the phosphorylation levels of ERK and STAT3 secondary to hypoxia was strongly associated with the attenuated NK cell cytotoxicity. Focusing on the mechanism responsible for reduced phosphorylation levels of ERK and STAT3, we reveal that the activation of protein tyrosine phosphatase SHP-1 (Src homology region 2 domain-containing phosphatase-1) following hypoxia might play an essential role in this process. By knocking down SHP-1 or blocking its activity using a specific inhibitor TPI-1, we were able to partially restore NK cell cytotoxicity under hypoxia. Taken together, we demonstrate that hypoxia could impair NK cell cytotoxicity by decreasing the phosphorylation levels of ERK and STAT3 in a SHP-1-dependent manner. Therefore, targeting SHP-1 could provide an approach to enhance NK cell-based tumor immunotherapy.

A novel CD2 staining-based flow cytometric assay for assessment of natural killer cell cytotoxicity.

BACKGROUND: Assessing cytotoxicity is fundamental to studying natural killer (NK) cell function. Various radioactive and non-radioactive cytotoxicity assays measuring target cell death have been developed. Among these methods, the most commonly used 51 Chromium-release assay (CRA) and flow cytometry-based cytotoxicity assays (FCCs) are the major representatives. Nonetheless, several drawbacks, including dye leakage and the potential effects of prior labeling on cells, curb the broad applicability of the FCCs. METHODS: Here, we report a rapid FCC for quantifying target cell death after co-incubation with NK cells. In this assay, after 4 hours of NK cell-target cell co-incubation, fluorochrome-conjugated CD2 antibody was used to identify NK cells, and SYTOX Green and Annexin V-FITC were further used to detect target cell death in CD2-negative population. In parallel, both CRA and FCC assay using CFSE/ 7-AAD were performed to validate the reproducibility and replicability. RESULTS: We observed that CD2 is exclusively positive on NK cells other than the most common hematological target tumor cells, such as K562, HL60, MOLM13, Raji, NCI-H929, rpmi8226, MM.1S, and KMS11. Assessment of target cell death using the CD2-based FCC shows a significantly higher percent specific lysis of the target cells compared to the standard CRA and the FCC assay using CFSE and 7-AAD. CONCLUSIONS: We demonstrated that this CD2-based FCC is a fast, simple, and reliable method for evaluating NK cell cytotoxicity.

Cell cycle exit during bortezomib-induced osteogenic differentiation of mesenchymal stem cells was mediated by Xbp1s-upregulated p21Cip1 and p27Kip1.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into a variety of cell types. Bortezomib, the first approved proteasome inhibitor used for the treatment of multiple myeloma (MM), has been shown to induce osteoblast differentiation, making it beneficial for myeloma bone disease. In the present study, we aimed to investigate the effects and underlying mechanisms of bortezomib on the cell cycle during osteogenic differentiation. We confirmed that low doses of bortezomib can induce MSCs towards osteogenic differentiation, but high doses are toxic. In the course of bortezomib-induced osteogenic differentiation, we observed cell cycle exit characterized by G0 /G1 phase cell cycle arrest with a significant reduction in cell proliferation. Additionally, we found that the cell cycle exit was tightly related to the induction of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1 . Notably, we further demonstrated that the up-regulation of p21Cip1 and p27Kip1 is transcriptionally dependent on the bortezomib-activated ER stress signalling branch Ire1α/Xbp1s. Taken together, these findings reveal an intracellular pathway that integrates proteasome inhibition, osteogenic differentiation and the cell cycle through activation of the ER stress signalling branch Ire1α/Xbp1s.

Blockade of HSP70 by VER-155008 synergistically enhances bortezomib-induced cytotoxicity in multiple myeloma.

Proteasome inhibitor bortezomib is one of the most effective drugs currently available for the treatment of multiple myeloma (MM). However, the intrinsic and acquired resistance to bortezomib can limit its effectiveness. The activation of heat shock response has been characterized as a potential resistance mechanism protecting MM cells from bortezomib-induced cell death. In this study, in response to bortezomib therapy, we discovered that HSP70 is one of the most substantially upregulated heat shock proteins. In order to further explore approaches to sensitizing bortezomib-based treatment for MM, we investigated whether targeting HSP70 using a specific inhibitor VER-155008 combined with bortezomib could overcome the acquired resistance in MM. We found that HSP70 inhibitor VER-155008 alone significantly decreased MM cell viability. Moreover, the combination of VER-155008 and bortezomib synergistically induced MM cell apoptosis markedly in vitro. Notably, the combined treatment was found to increase the cleavage of PARP, an early marker of chemotherapy-induced apoptosis. Importantly, the reduction of anti-apoptotic Bcl-2 family member Bcl-2, Bcl-xL, and Mcl-1 and the induction of pro-apoptotic Bcl-2 family member BH3-only protein NOXA and Bim were confirmed to be tightly associated with the synergism. Finally, the ER stress marker CHOP (CCAAT-enhancer binding protein homologous protein), which can cause transcriptional activation of genes involved in cell apoptosis, was markedly induced by both VER-155008 and bortezomib. Taken together, our finding of a strong synergistic interaction between VER-155008 and bortezomib may support for combination therapy in MM patients in the future.

Hypoxia promotes osteosarcoma cell proliferation and migration through enhancing platelet-derived growth factor-BB/platelet-derived growth factor receptor-ß axis.

Osteosarcoma is a primary malignant bone tumor, characterized by high therapeutic resistance and poor outcomes, due to unclear pathological mechanisms. It has been shown recently that the platelet-derived growth factor (PDGF)/platelet-derived growth factor receptor (PDGFR) pathway is closely associated with the pathogenesis of osteosarcoma. Hypoxia is a critical hallmark of tumor microenvironment that promotes the malignant phenotype in many solid tumors and a fundamental impediment to effective tumor therapy. In this study, we confirmed that hypoxia is an important feature of osteosarcoma, validated by the positive immunohistochemistry staining of hypoxia marker hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase IX (CAIX) in osteosarcoma tissue samples. More importantly, we discovered that hypoxia could transcriptionally upregulate the expression of both PDGF-BB and PDGFR-ß in osteosarcoma cells in vitro. Likewise, we also established that hypoxia-induced PDGF-BB is strongly related to the enhanced cell proliferation and migration, by activating AKT, ERK1/2, and STAT3 signaling pathways. Notably, when using an antibody to block the autocrine of PDGF-BB, cell proliferation and migration were partially aborted in hypoxia. Collectively, we demonstrated that the hypoxia-activated PDGF-BB/PDGFR-ß axis plays essential roles in osteosarcoma progression. These findings may shed light on the molecular pathogenesis of osteosarcoma, and provide a novel strategy for osteosarcoma treatment by combinational targeting hypoxia and PDGF-BB/PDGFR signaling.

The influence of chitosan hydrogel on stem cell engraftment, survival and homing in the ischemic myocardial microenvironment.

One challenge of cellular cardiomyoplasty for myocardial infarction (MI) is how to improve MI microenvironment to facilitate stem cell engraftment, survival and homing for myocardial repair. The application of injectable hydrogels is an effective strategy. However, it has not been thoroughly investigated on the role of the injectable scaffolds, in improving MI microenvironment, providing space and guidance for cell survival, engraftment and homing. We explored an injectable chitosan hydrogel for stem cell delivery into ischemic heart and investigated the beneficial effects and mechanisms of the hydrogel. In vitro, H(2)O(2)-treatment was used to mimic reactive oxygen species (ROS) microenvironment. The influence of ROS and protection of chitosan components on adipose-derived mesenchymal stem cells (ADSCs) was analyzed too. In vivo, MI was induced by the left anterior descending artery ligation in SD rats. PBS, chitosan hydrogel, ADSC/PBS and ADSC/chitosan hydrogel were injected into the border of infracted hearts respectively. Multi-techniques were used to assess the beneficial effects of chitosan hydrogel after transplantation. We observed that ROS generated by ischemia would impair ADSC adhesion molecules, including integrin-related adhesion molecules integrin αV and ß1, focal adhesion-related molecules p-FAK and p-Src, and corresponding ligands of host myocardium ICAM1 and VCAM1. Chitosan hydrogel could rescue these molecules through ROS scavenging and recruit key chemokine for stem cell homing, such as SDF-1. The results suggest that chitosan hydrogel could improve MI microenvironment, enhance stem cell engraftment, survival and homing in ischemic heart through ROS scavenging and chemokine recruitment, contributing to myocardial repair.

Tumourigenesis in the infarcted rat heart is eliminated through differentiation and enrichment of the transplanted embryonic stem cells.

AIMS: The therapeutic potential of embryonic stem cells (ESCs) in ischaemic heart disease has been widely explored. However, tumourigenesis upon implantation interferes with the clinical application of ESC transplantation. This study aims to evaluate the influence of differentiation and enrichment of transplanted ESCs on tumourigenesis in infarcted rat hearts. METHODS AND RESULTS: Mouse ESCs (mESCs) were cultured using a bioreactor system to develop embryoid bodies, which were then induced with 1% ascorbic acid to differentiate into cardiomyocytes. The mESCs-derived cardiomyocytes (mESCs-CMs) were enriched by Percoll density gradient separation. The specific markers (OCT-4, Sox2, and Nanog) of undifferentiated ESCs were detected by PCR both in mESCs and in mESCs-CMs, but not in the mESC-derived Percoll-enriched cardiomyocytes (mESC-PE-CMs). Immunosuppressed rats with infarcted hearts were randomly injected with the mESCs, mESC-CMs, or mESC-PE-CMs. Eight weeks after cell transplantation, histological and immunohistochemical analysis showed that the transplantation of both mESCs and mESC-CMs caused the formation of teratomas. The incidence of teratoma was markedly lower (P < 0.05) in the mESC-CMs group than in the mESCs group. The average tumour volume was significantly lower (P < 0.05) in the mESC-CMs group than in the mESCs group. Tumour formation was absent in the mESC-PE-CMs group. CONCLUSION: Enrichment of the mESC-differentiated cardiomyocytes inhibited the development of teratoma after cell transplantation in the infarcted rat hearts. These findings offer a new strategy for eliminating teratoma formation in ESCs transplantation and could be a step forward in the development of human ESCs transplantation therapy in ischaemic heart disease.

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds.

Embryonic stem (ES) cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy. The main catalyst for ES cells differentiation is the growth into embryoid bodies (EBs), which are utilized widely as the trigger of in vitro differentiation. In this study, a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established. When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds, they grew into aggregates gradually and formed simple EBs with circular structures. After 7 days' culture, they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers. Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types; they were also able to form into tissue-like structures. Moreover, with introduction of ascorbic acid, ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19. The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment.

Improved myocardial performance in infarcted rat heart by co-injection of basic fibroblast growth factor with temperature-responsive chitosan hydrogel.

BACKGROUND: Basic fibroblast growth factor (bFGF) stimulates neoangiogenesis. The sustained release of bFGF by using biomaterials helped to enhance its angiogenic activity in vivo. In this study we investigated the effects of co-injection of bFGF with temperature-responsive chitosan hydrogel on myocardial performance in a rat model of infarction. METHODS: Myocardial infarction was induced in rats using coronary artery ligation. Temperature-responsive chitosan hydrogel was prepared and injected intramyocardially into the left ventricular wall of rat infarction models alone or together with bFGF. Detailed histologic analysis and echocardiography were used to determine the structural and functional consequences 4 weeks after injection. RESULTS: Heart function improved significantly in the chitosan+bFGF group compared with the phosphate-buffered saline (PBS)+bFGF group with regard to left ventricular ejection fraction (LVEF) and LV fractional shortening (LVFS) 4 weeks after transplantation (p < 0.05, n = 8 per group). In addition, arteriole densities within the infarcted area improved significantly (p < 0.01) in the chitosan+bFGF group (259 +/- 22/mm(2)) compared with the PBS+bFGF group (95 +/- 18/mm(2); n = 8 per group) at 4 weeks after transplantation. Infarct size and fibrotic area decreased significantly (p < 0.05) in the chitosan+bFGF group (39.64 +/- 1.75% and 25.09 +/- 3.31%, respectively) compared with the PBS+bFGF group (48.91 +/- 1.39% and 48.0 +/- 3.83%, respectively; n = 8 per group). No significant difference (p > 0.05) was noted between the PBS and PBS+bFGF groups. CONCLUSIONS: Co-injection of bFGF with temperature-responsive chitosan hydrogels enhanced the effects of bFGF on arteriogenesis, ventricular remodeling and cardiac function. Our findings suggest a new approach to improve infarcted repairs in the prevention of adverse remodeling after myocardial infarction.

Efficient isolation of cardiac stem cells from brown adipose.

Cardiac stem cells represent a logical cell type to exploit in cardiac regeneration. The efficient harvest of cardiac stem cells from a suitable source would turn promising in cardiac stem cell therapy. Brown adipose was recently found to be a new source of cardiac stem cells, instrumental to myocardial regeneration. Unfortunately, an efficient method for the cell isolation is unavailable so far. In our study we have developed a new method for the efficient isolation of cardiac stem cells from brown adipose by combining different enzymes. Results showed that the total cell yield dramatically increased (more than 10 times, P < .01) compared with that by previous method. The content of CD133-positive cells (reported to differentiate into cardiomyocytes with a high frequency) was much higher than that in the previous report (22.43% versus 3.5%). Moreover, the isolated cells could be the efficiently differentiated into functional cardiomyocytes in optimized conditions. Thus, the new method we established would be of great use in further exploring cardiac stem cell therapy.

Both the transplantation of somatic cell nuclear transfer- and fertilization-derived mouse embryonic stem cells with temperature-responsive chitosan hydrogel improve myocardial performance in infarcted rat hearts.

The transplantation of embryonic stem cells could improve cardiac function but was limited by immune rejection as well as low cell retention and survival within the ischemic tissues. The somatic cell nuclear transfer (SCNT) is practical to generate autologous histocompatible stem (nuclear-transferred embryonic stem [NTES]) cells for diseases, but NTES may be arguably unsafe for therapeutic application. The temperature-responsive chitosan hydrogel is a suitable matrix in cell transplantation. As the scaffold, chitosan hydrogel was coinjected with NTES cells into the left ventricular wall of rat infarction models. Detailed histological analysis and echocardiography were performed to determine the structure and functional consequences of transplantation. The myocardial performance in SCNT- and fertilization-derived mouse ES cell transplantation with chitosan hydrogel was also compared. The results showed that both the 24-h cell retention and 4-week graft size were significantly greater in the NTES + chitosan group than that of NTES + phosphate-buffered saline (PBS) group (p < 0.01). The NTES cells might differentiate into cardiomyocytes in vivo. The heart function improved significantly in the chitosan + NTES group (fractional shortening: 28.7% +/- 2.8%) compared with that of PBS + NTES group (fractional shortening: 25.2% +/- 2.9%) at 4 weeks after transplantation (p < 0.01). In addition, the arteriole/venule densities within the infarcted area improved significantly in the chitosan + NTES group (280 +/- 17/mm(2)) compared with that of PBS + NTES group (234 +/- 16/mm(2)) at 4 weeks after transplantation (p < 0.01). There was no difference in the myocardial performance in SCNT- and fertilization-derived mouse ES cell transplantation with chitosan hydrogel. The NTES cells with chitosan hydrogel have been proved to possess therapeutic potential to improve the function of infarcted heart. Thus the method of in situ injectable tissue engineering is promising clinically.

Bioreactor cultivation enhances NTEB formation and differentiation of NTES cells into cardiomyocytes.

Autogenic embryonic stem cells established from somatic cell nuclear transfer (SCNT) embryos have been proposed as unlimited cell sources for cell transplantation-based treatment of many genetic and degenerative diseases, which can eliminate the immune rejection that occurs after transplantation. In the present study, pluripotent nuclear transfer ES (NTES) cell lines were successfully established from different strains of mice. One NTES cell line, NT1, with capacity of germline transmission, was used to investigate in vitro differentiation into cardiomyocytes. To optimize differentiation conditions for mass production of embryoid bodies (NTEBs) from NTES cells, a slow-turning lateral vessel (STLV) rotating bioreactor was used for culturing the NTES cells to produce NTEBs compared with a conventional static cultivation method. Our results demonstrated that the NTEBs formed in STLV bioreactor were more uniform in size, and no large necrotic centers with most of the cells in NTEBs were viable. Differentiation of the NTEBs formed in both the STLV bioreactor and static culture into cardiomyocytes was induced by ascorbic acid, and the results demonstrated that STLV-produced NTEBs differentiated into cardiomyocytes more efficiently. Taken together, our results suggested that STLV bioreactor provided a more ideal culture condition, which can facilitate the formation of better quality NTEBs and differentiation into cardiomyocytes more efficiently in vitro.