A rapid degradation of calponin 2 is required for cytokinesis.

Calponin 2 is an actin cytoskeleton-associated protein and plays a role in regulating cell motility-related functions such as phagocytosis, migration, and division. We previously reported that overexpression of calponin 2 inhibits the rate of cell proliferation. To investigate the underlying mechanism, our present study found that the levels of endogenous calponin 2 in NIH3T3 and HEK293 cells rapidly decreased before cell division characterized by an absence at the actin contractile ring. In cells lacking endogenous calponin 2, transfective expression of GFP-fusion calponin 2 inhibited cell proliferation similar to that of nonfusion calponin 2. Fluorescent imaging studies of mitotic cells indicated that a proper level of calponin 2 expression and effective degradation during cytokinesis are necessary for normal cell division. Computer-assisted dynamic image analysis of dividing cells revealed that overexpression of calponin 2 significantly affects motility and shape behaviors of cells only on the interval from the start of anaphase to the start of cytokinesis, i.e., the pre-cytokinesis phase, but not on the interval from the start of cytokinesis to 50% completion of cytokinesis. The pre-cytokinesis degradation of calponin 2 was attenuated by MG132 inhibition of the ubiquitin proteasome and inhibitor of protein kinase C (PKC), suggesting that PKC phosphorylation-triggered degradation of calponin 2 could determine the rate of cytokinesis. The novel role of calponin 2 in regulating the rate of cytokinesis may be targeted for therapeutic applications such as in an inhibition of malignant tumor growth.

Does the endometrial receptivity array really provide personalized embryo transfer?

PURPOSE: The aim of the present study was to determine the percentage of infertility patients who are diagnosed with a non-receptive endometrium according to the endometrial receptivity array (ERA) test and to examine whether adjusting the embryo transfer day according to the proposed shift in the window of implantation improves the pregnancy rate compared to non-ERA-tested patients. METHODS: A single-center retrospective cohort study, including 53 consecutive good prognosis patients (0-2 previous frozen embryo transfers) admitted to our IVF unit for a mock cycle prior to their frozen day-5 embryo (blastocyst) transfer cycle. The mock cycle included an endometrial biopsy for both the ERA test and histological assessment by the Noyes criteria (study group). The next cycle frozen embryo transfer (FET) in the study group was adjusted according to the ERA results. The control group consisted of patients who underwent FET cycles at our clinic during the same period, without performing the endometrial biopsy and ERA testing. RESULTS: During the study period, 503 patients (control group) underwent FET cycles without performing the ERA testing and 41 patients had FET following an ERA test. There were no between-group differences in patients' age, number of previous transfers, endometrial thickness, number of transferred embryos, and ongoing pregnancy rates (35.2 vs. 39%, respectively, p = NS). Out of the 53 patients who performed the ERA test before their first or second FET, five endometrial samples (9.4%) were found to be post-receptive, 29 (54.7%) pre-receptive, and only 19 samples (35.8%) were receptive. Women in the study group with pre- or post-receptive endometrium on ERA testing, the appropriate adjustment in timing of FET according to the ERA test resulted in a 33.3% pregnancy rate, which is comparable to the 35.2% background ongoing pregnancy rate of the control group. CONCLUSIONS: Performing the ERA test in a mock cycle prior to a FET does not seem to improve the ongoing pregnancy rate in good prognosis patients. Further large prospective studies are needed to elucidate the role of ERA testing in both good prognosis patients and in patients with recurrent implantation failure.

Recovery of CD45(-)/Lin(-)/SSEA-4(+) very small embryonic-like stem cells by cord blood bank standard operating procedures.

BACKGROUND AIMS: Very small embryonic-like (VSEL) stem cells are a rare cell population present in bone marrow, cord blood and other tissues that displays a distinct small cell size and the ability to give rise to cells of the three germ layers. VSEL stem cells were reported to be discarded in the red blood cell fraction by Ficoll-Paque density gradient centrifugation during the processing of bone marrow and cord blood specimens. However, most cord blood banks do not include density gradient centrifugation in their procedures while red blood cells are removed by Hespan sedimentation following the Cord Blood Transplantation Study cord blood bank standard operating procedures (COBLT SOP). To clarify the retention of VSEL stem cells, we investigated the recovery of VSEL stem cells following COBLT SOP guidelines. METHODS: The recovery of CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells of umbilical cord blood was examined by flow cytometry before and after COBLT SOP processing, and relative expression of pluripotent genes was analyzed by quantitative polymerase chain reaction. RESULTS: CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells were mostly recovered in the final products following COBLT SOP guidelines. The expression of pluripotent genes could be maintained at >80% in products after hetastarch (Hespan; B. Braun Medical Inc., Irvine, CA, USA) processing. CONCLUSIONS: The rare sub-population of CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells survived after Hespan sedimentation. This finding suggests that umbilical cord blood units cryopreserved by COBLT SOP in cord blood banks should retain most VSEL stem cells present in the un-processed specimens.

Loss of Calponin 2 causes premature ovarian insufficiency in mice.

BACKGROUND: Premature ovarian insufficiency (POI) is a condition defined as women developing menopause before 40 years old. These patients display low ovarian reserve at young age and difficulties to conceive even with assisted reproductive technology. The pathogenesis of ovarian insufficiency is not fully understood. Genetic factors may underlie most of the cases. Actin cytoskeleton plays a pivotal role in ovarian folliculogenesis. Calponin 2 encoded by the Cnn2 gene is an actin associated protein that regulates motility and mechanical signaling related cellular functions. RESULTS: The present study compared breeding of age-matched calponin 2 knockout (Cnn2-KO) and wild type (WT) mice and found that Cnn2-KO mothers had significantly smaller litter sizes. Ovaries from 4 weeks old Cnn2-KO mice showed significantly lower numbers of total ovarian follicles than WT control with the presence of multi-oocyte follicles. Cnn2-KO mice also showed age-progressive earlier depletion of ovarian follicles. Cnn2 expression is detected in the cumulus cells of the ovarian follicles of WT mice and colocalizes with actin stress fiber, tropomyosin and myosin II in primary cultures of cumulus cells. CONCLUSIONS: The findings demonstrate that the loss of calponin 2 impairs ovarian folliculogenesis with premature depletion of ovarian follicles. The role of calponin 2 in ovarian granulosa cells suggests a molecular target for further investigations on the pathogenesis of POI and for therapeutic development.

Evolution and function of calponin and transgelin.

Calponin and transgelin (originally named SM22) are homologous cytoskeleton proteins that regulate actin-activated myosin motor functions in smooth muscle contraction and non-muscle cell motility during adhesion, migration, proliferation, phagocytosis, wound healing, and inflammatory responses. They are abundant cytoskeleton proteins present in multiple cell types whereas their physiological functions remain to be fully established. This focused review summarizes the evolution of genes encoding calponin and transgelin and their isoforms and discusses the structural similarity and divergence in vertebrate and invertebrate species in the context of functions in regulating cell motility. As the first literature review focusing on the evolution of the calponin-transgelin family of proteins in relevance to their structure-function relationship, the goal is to outline a foundation of current knowledge for continued investigations to understand the biological functions of calponin and transgelin in various cell types during physiological and pathological processes.

Deletion of Calponin 2 Reduces the Formation of Postoperative Peritoneal Adhesions.

Aim of the study: Postoperative peritoneal adhesions are a common cause of morbidity after surgery, resulting in multiple complications. Macrophage-mediated inflammation and myofibroblast differentiation after tissue injury play central roles in the pathogenesis and progression of adhesion formation. Calponin 2 is an actin cytoskeleton regulatory protein in endothelial cells, macrophages and fibroblasts that are key players in the development of fibrosis. Deletion of calponin 2 has been shown to attenuate inflammatory arthritis, atherosclerosis and fibrocalcification of the aortic valves. The present study investigated the effect of calponin 2 deletion on attenuating the formation of peritoneal adhesions in a mouse model for potential use as a new therapeutic target.Materials and methods: Sterile surgical procedures under general anesthesia were used on paired wild type (WT) and calponin 2 knockout (KO) mice to generate mild injury on the cecal and abdominal wall peritonea. Three and seven days post-operation, the mice were compared postmortem for the formation of peritoneal adhesions. Tissues at the adhesion sites were examined with histology and immunofluorescent studies for macrophage and myofibroblast activations.Results: Quantitative scoring demonstrated that calponin 2 KO mice developed significantly less postoperative peritoneal adhesions than that in WT mice. Calponin 2 deletion resulted in less infiltration of F4/80+ macrophages at the adhesion sites with less myofibroblast differentiation and collagen deposition than WT controls.Conclusions: The data show that deletion of calponin 2 effectively reduces postoperative peritoneal adhesion, presenting a novel molecular target for clinical prevention.

Loss of Calponin 2 causes age-progressive proteinuria in mice.

Proteinuria is a major manifestation of kidney disease, reflecting injuries of glomerular podocytes. Actin cytoskeleton plays a pivotal role in stabilizing the foot processes of podocytes against the hydrostatic pressure of filtration. Calponin is an actin associated protein that regulates mechanical tension-related cytoskeleton functions and its role in podocytes has not been established. Here we studied the kidney phenotypes of calponin isoform 2 knockout (KO) mice. Urine samples were examined to quantify the ratio of albumin and creatinine. Kidney tissue samples were collected for histology and ultrastructural studies. A mouse podocyte cell line (E11) was used to study the expression and cellular localization of calponin 2. In comparison with wild-type (WT) controls, calponin 2 KO mice showed age-progressive high proteinuria and degeneration of renal glomeruli. High levels of calponin 2 are expressed in E11 podocytes and colocalized with actin stress fibers, tropomyosin and myosin IIA. Electron microscopy showed that aging calponin 2 KO mice had effacement of the podocyte foot processes and increased thickness of the glomerular basement membrane as compared to that of WT control. The findings demonstrate that deletion of calponin 2 aggravates age-progressive degeneration of the glomerular structure and function as filtration barrier. The critical role of calponin 2 in podocytes suggests a molecular target for understanding the pathogenesis of proteinuria and therapeutic development.

Large generation of megakaryocytes from serum-free expanded human CD34+ cells.

Ex vivo generation of megakaryocytes from hematopoietic stem cells (HSCs) is crucial to HSC research and has important clinical potential for thrombocytopenia patients to rapid platelet reconstruction. In this study, factorial design and steepest ascent method were used to screen and optimize the effective cytokines (10.2 ng/ml TPO, 4.3 ng/ml IL-3, 15.0 ng/ml SCF, 5.6 ng/ml IL-6, 2.8 ng/ml FL, 2.8 ng/ml IL-9, and 2.8 ng/ml GM-CSF) in megakaryocyte induction medium that facilitate ex vivo megakaryopoiesis from CD34(+) cells. After induction, the maximum fold expansion for accumulated megakaryocytes was almost 5000-fold, and the induced megakaryocytes were characterized by analysis of gene expression, polyploidy and platelet activation ability. Furthermore, the combination of megakaryocyte induction medium and HSC expansion medium can induce and expand a large amount of functional megakaryocytes efficiently, and might be a promising source of megakaryocytes and platelets for cell therapy in the future.

Chondrogenic differentiation of human mesenchymal stem cells from umbilical cord blood in chemically synthesized thermoreversible polymer.

Scaffolds provide a template for cell distribution, growth, differentiation and extracellular matrix accumulation in a three-dimensional environment. Recent studies have demonstrated the potential of scaffolds for enhancing articular cartilage repair both in vitro and in vivo investigations. Mesenchymal stem cells derived from human umbilical cord blood (CBMSCs) have been characterized by their multipotency to differentiate into mesenchyme-lineage cell types, including chondrocytes, osteoblasts, and adipocytes. In this study, chondrogenesis of CBMSCs was performed in a chemically synthesized thermoreversible gelation polymer (TGP). CBMSCs were embedded in the TGP and supplemented with ascorbic acid and transforming growth factor-beta 3. After a 4-week induction, the results showed that CBMSCs formed into spheroid pellets and increased in size. The induced cells in the TGP expressed specific mRNA of collagen type II, aggrecan, and Sox9 for chondrocytes. Furthermore, CBMSCs embedded in TGP had higher ratio of glycosaminoglycan secretion to DNA content than the traditional induction method by aggregating pellet culture. These results demonstrated that chemically synthesized TGP provided a competent 3-dimentional culture environment for CBMSCs to differentiate into chondrocytes and may be applied clinically to induce chondrogenic differentiation of CBMSCs for cartilage repair in the future.

Recent advances in in vitro fertilization.

The field of assisted reproductive technology is rapidly progressing with many new advances in the last decade. The present review discusses methods to improve oocyte quality in older women and new stimulation protocols that may improve the number of mature oocytes retrieved during an in vitro fertilization cycle. We will discuss the present use of pre-implantation genetic screening (PGS) and finally focus on some new methods to determine endometrial receptivity. The focus of this review is to point out areas of technology that may be controversial or are new enough to require proper controlled studies for validation.

Characterization of serum-free ex vivo-expanded hematopoietic stem cells derived from human umbilical cord blood CD133(+) cells.

The development of ex vivo expansion of hematopoietic stem cells (HSCs) is a promising approach to restore the required bone marrow function of patients with hematological disorders. Previously, we have reported the development of an optimized serum-free and cytokines-limited defined medium using statistic methodology for umbilical cord blood-derived HSC expansion. The aim of this study was to analyze further the characteristics and functions of cells in vitro and in vivo when cultured in this defined medium. After a 7-day batch culture, the average absolute fold expansions for CD133(+) cells, CD34(+)CD133(+) cells, CD34(+)CD38() cells, CD133(+)CD38(-) cells, CD34(+)CXCR4(+) cells, CD133(+)CXCR4(+) cells, and long-term culture-initiating cells were 21-, 20-, 723-, 618-, 160-, 384-, and 8-fold, respectively. The high enrichment of CD38(-) cells and CXCR4(+) cells of the CD34(+) subpopulation provided a very early uncommitted HSC proliferation and homing ability. Furthermore, the expanded cells showed a high level of telomerase activity to maintain their telomere length and repopulated the lethally irradiated NOD/SCID mice in vivo. These results indicated that the cytokines limited expanded cells from CD133(+) cells could substantially support simultaneous expansion of various stem/progenitor cells and engraft with the expanded cells from a low number of HSCs initially.

A systematic strategy to optimize ex vivo expansion medium for human hematopoietic stem cells derived from umbilical cord blood mononuclear cells.

OBJECTIVE: In this study, a serum-free, stroma-free, and chemically defined medium for hematopoietic stem cell (HSC) expansion was systematically developed and optimized using factorial design and the steepest ascent method. MATERIALS AND METHODS: Mononuclear cells (MNCs) were isolated from umbilical cord blood (UCB). HSCs were stimulated to proliferate ex vivo in the MNC culture system with variable serum substitutes, cytokines, and basal media according to experimental design. The expanded cells were assessed for cellular characteristics by surface antigen analysis, colony-forming cell assay (CFC assay), and long-term culture-initiating cell assay (LTC-IC assay). RESULTS: The optimal compositions of serum substitutes and the cytokine cocktail for HSC expansion in the MNC culture system were BIT (4 g/L BSA, 0.71 microg/mL insulin, and 27.81 microg/mL transferrin), and CC-9 (5.53 ng/mL TPO, 2.03 ng/mL IL-3, 16 ng/mL SCF, 4.43 ng/mL FL, 2.36 ng/mL IL-6, 1.91 ng/mL G-CSF, 1.56 ng/mL GM-CSF, 2.64 ng/mL SCGF, and 0.69 ng/mL IL-11) in the Iscove's modified Dulbecco's medium. After 6-day culture, the absolute fold expansions for white blood cells, CD34+ cells, CD34+CD38- cells, CFC, and LTC-IC were 1.4-, 30.4-, 63.9-, 10.7-, 2.8-fold, respectively. CONCLUSION: Using the statistic methodology to develop HSC medium, our formula had lower cytokine concentrations comparing to other literatures and commercial media, but had superior or comparable expansion ability on HSC growth.

Characterization and transplantation of induced megakaryocytes from hematopoietic stem cells for rapid platelet recovery by a two-step serum-free procedure.

OBJECTIVE: A complete process for mass generation of megakaryocytes from hematopoietic stem cells under serum-free conditions has great clinical potential for rapid platelet reconstruction in thrombocytopenia patients. We have previously reported on the generation of an optimized serum-free medium (serum-free hematopoietic stem cell medium) for ex vivo expansion of CD34(+) cells. Here, we further generated large amounts of functional megakaryocytes from serum-free expanded CD34(+) cells under a complete and optimal serum-free condition for complying with clinical regulations. MATERIALS AND METHODS: Serum substitutes and cytokines were screened and optimized for their concentration for megakaryocyte generation by systemically methods. Serum-free induced megakaryocytes were characterized by surface antigens, gene expression, ex vivo megakaryocyte activation ability, and ability of megakaryocyte and platelet recovery in nonobese diabetic/severe combined immunodeficient mice. RESULTS: The optimal serum-free megakaryocyte induction medium was Iscove's modified Dulbecco's medium containing serum substitutes (i.e., human serum albumin, human insulin, and human transferrin) and a cytokine cocktail (i.e., thrombopoietin, stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor). After induction, induced megakaryocytes expressed CD41a and CD61 surface antigens, nuclear factor erythroid-derived 2 and GATA-1 transcription factors and megakaryocyte activation ability. Importantly, transplantation of induced megakaryocytes could accelerate megakaryocyte and platelet recovery in irradiated nonobese diabetic/severe combined immunodeficient mice. CONCLUSION: In conclusion, we have developed a serum-free megakaryocyte induction medium, and the combination of serum-free megakaryocyte and serum-free hematopoietic stem cell media can generate a large amount of functional megakaryocytes efficiently. Our method represents a promising source of megakaryocytes and platelets for future cell therapy.

Characterization of two populations of mesenchymal progenitor cells in umbilical cord blood.

Umbilical cord blood (UCB) is a valuable source for hematopoietic progenitor cell therapy. Moreover, it contains another subset of non-hematopoietic population referred to as mesenchymal progenitor cells (MPCs), which can be ex vivo expanded and differentiated into osteoblasts, chondrocytes and adipocytes. In this study, we successfully isolated the clonogenic MPCs from UCB by limiting dilution method. These cells exhibited two different morphologic phenotypes, including flattened fibroblasts (majority) and spindle-shaped fibroblasts (minority). Both types of MPCs shared similar cell surface markers except CD90 and had similar osteogenic and chondrogenic potentials. However, the spindle-shaped clones possessed the positive CD90 expression and showed a greater tendency in adipogenesis, while the flattened clones were CD90 negative cells and showed a lower tendency in adipogenesis. The high number of flattened MPCs might be linked to the less sensitivity of UCB-derived MPCs in adipogenic differentiation.

Disparate mesenchyme-lineage tendencies in mesenchymal stem cells from human bone marrow and umbilical cord blood.

Bone marrow and umbilical cord blood are reported to be the main sources of mesenchymal stem cells (MSCs), which have been proposed for many clinical applications. This study evaluated and quantitated the differentiation potential of bone marrow-derived MSCs (bmMSCs) and cord blood-derived MSCs (cbMSCs) by in vitro induction. Results indicated that cbMSCs had a significantly stronger osteogenic potential but lower capacity for adipogenic differentiation than bmMSCs. Leptin, an important regulator of mesenchymal differentiation, has a significantly stronger effect of promoting osteogenesis and inhibiting adipogenesis in bmMSCs than in cbMSCs. Moreover, Cbfa1 mRNA expression in bmMSCs and cbMSCs was affected to different degrees by leptin during osteogenesis. In contrast, leptin reduced PPARgamma2 mRNA expression to the same level during adipogenesis in both types of MSCs. These results demonstrate the disparate capacities of MSCs from bone marrow and cord blood and suggest that they be used differently in experimental and therapeutic studies. In addition, the disparate differentiation tendencies of MSCs from different sources should be considered in further applications.