Rho-Associated Kinase Inhibitor (Y-27632) Attenuates Doxorubicin-Induced Apoptosis of Human Cardiac Stem Cells

Background

Recent clinical trials using c-kit⁺ human cardiac stem cells (CSCs) demonstrated promising results in increasing cardiac function and improving quality of life. However, CSC efficiency is low, likely due to limited cell survival and engraftment after transplantation. The Rho-associated protein kinase (ROCK) inhibitor, Y-27632, significantly increased cell survival rate, adhesion, and migration in numerous types of cells, including stem cells, suggesting a common feature of the ROCK-mediated apoptotic pathway that may also exist in human CSCs. In this study, we examine the hypothesis that pretreatment of human CSCs with Y-27632 protects cells from Doxorubicin (Dox) induced apoptosis.

Methods and Results

c-kit⁺ CSCs were cultured in CSC medium for 3–5 days followed by 48hr treatment with 0 to 10μM Y-27632 alone, 0 to 1.0μM Dox alone, or Y-27632 followed by Dox (48hrs). Cell viability, toxicity, proliferation, morphology, migration, Caspase-3 activity, expression levels of apoptotic-related key proteins and c-kit⁺ were examined. Results showed that 48hr treatment with Y-27632 alone did not result in great changes in c-kit⁺ expression, proliferation, Caspase-3 activity, or apoptosis; however cell viability was significantly increased and cell migration was promoted. These effects likely involve the ROCK/Actin pathways. In contrast, 48hr treatment with Dox alone dramatically increased Caspase-3 activity, resulting in cell death. Although Y-27632 alone did not affect the expression levels of apoptotic-related key factors (p-Akt, Akt, Bcl-2, Bcl-xl, Bax, cleaved Caspase-3, and Caspase-3) under basal conditions, it significantly inhibited the Dox-induced increase in cleaved Caspase-3 and reduced cell death under Dox treatment.

Conclusions

We conclude that preconditioning human CSCs with Y-27632 significantly reduces Dox-induced cell death and possibly involves the cleaved Caspase-3 and ROCK/Actin pathways. The beneficial effects of Y-27632 may be applied to stem cell-based therapy to increase cell survival rates after transplantation or to act as a cardiac protective agent for Dox-treated cancer patients.     

ROCK Inhibitor Is Not Required for Embryoid Body Formation from Singularized Human Embryonic Stem Cells

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.     

人类及小鼠胚胎干细胞的不同多能性状态

胚胎干细胞(embryonic stem cells,ESCs)是来源于早期胚胎的全能性细胞,在合适条件下具有分化为任何一类成体细胞的潜力。在小鼠中,根据细胞来源的胚胎发育时间,ESCs可以被分为原始态多能性(na(?)ve pluripotency)和始发态多能性(primed pluripotency)两种状态。这两种状态的细胞在发育上相互联系,具有不同的形态、信号依赖、发育性质、基因表达及表观遗传学性质,并且在特定的条件下可以相互转化。人类胚胎干细胞(human embryonic stem cells,hESCs)的发育潜能曾一度被认为低于小鼠胚胎干细胞(mouse embryonic stem cells,mESCs),直到人类原始态胚胎干细胞的发现证明了hESCs可以表现出与mESCs相似的性质。这对于人类胚胎发育的研究及ESCs在临床治疗上的实际应用都具有重要的意义。     

Caspase Inhibitor Z-VAD-FMK Enhances the Freeze-Thaw Survival Rate of Human Embryonic Stem Cells

Previous study demonstrated that the low survival of human embryonic stem cells (hESC) under conventional slow-cooling cryopreservation protocols is predominantly due to apoptosis rather than cellular necrosis. Hence, this study investigated whether a synthetic broad-spectrum irreversible inhibitor of caspase enzymes, Z-VAD-FMK can be used to enhance the post-thaw survival rate of hESC. About 100 mM Z-VAD-FMK was supplemented into either the freezing solution, the post-thaw culture media or both. Intact and adherent hESC colonies were cryopreserved so as to enable subsequent quantitation of the post-thaw cell survival rate through the MTT assay, which can only be performed with adherent cells. Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhance the post-thaw survival rate (10.2% vs. 9.9%, p > 0.05). However, when 100 mM Z-VAD-FMK was added to the post-thaw culture media, there was a significant enhancement in the survival rate from 9.9% to 14.4% (p < 0.05), which was further increased to 18.7% when Z-VAD-FMK was also added to the freezing solution as well (p < 0.01). Spontaneous differentiation of hESC after cryopreservation was assessed by morphological observations under bright-field microscopy, and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. The results demonstrated that exposure to Z-VAD-FMK did not significantly enhance the spontaneous differentiation of hESC within post-thaw culture.     

Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

Human embryonic stem cells have the ability to generate all cell types in the body and can potentially provide an unlimited source of cells for cell replacement therapy to treat degenerative diseases such as diabetes. Current differentiation protocols of human embryonic stem cells towards insulin producing beta cells focus on soluble molecules whereas the impact of cell-matrix interactions has been mainly unattended. In this study almost 500 different extracellular matrix protein combinations were screened to systemically identify extracellular matrix proteins that influence differentiation of human embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed by time lapse studies compared to cells on the other tested substrates. Global gene expression analysis showed that cells differentiated on collagen I were largely similar to cells on fibronectin after completed differentiation. Collectively, the data suggest that collagen I induces a more rapid and consistent differentiation of stem cells to definitive endoderm. The results shed light on the importance of extracellular matrix proteins for differentiation and also points to a cost effective and easy method to improve differentiation.     

免疫磁珠分选人角朊干细胞的实验研究

目的:探索人角朊干细胞(keratinocyte stem cells,KSCs)的免疫磁珠分选(Magnetic Activated Cell Sorting,MACS)的方法。方法:从人包皮组织获得角朊细胞悬液,然后利用人角朊干细胞表面CD49f(整合素α6)和CD71的表达情况(CD49fbriCD71dim)通过MACS法将KSCs分选出来,在荧光显微镜下观察其荧光标记情况,同时将分选所得的CD49fbriCD71dim细胞进行体外无血清培养,观察其形态及克隆形成。结果:人角朊细胞经MACS法分选后所得的CD49fbriCD71dim细胞比率可达12．02(±6．92)%。CD49fbriCD71dim细胞经培养可见细胞为典型的上皮样特征,呈铺路石样形态,高核浆比例,细胞紧密排列,轮廓清楚,折光性好,并可在5-7天左右形成全克隆,符合KSCs的特点。结论:研究表明MACS分选法可以得到较高比例的人角朊干细胞,并能进行后续培养研究,其不失为一种较理想的人角朊干细胞的分选方法。     

Comparison of Surface Markers between Human and Rabbit Mesenchymal Stem Cells

This study investigated whether there are marked differences in surface markers between rabbit and human mesenchymal stem cells (MSCs). Murine and rabbit MSCs have been reported to be CD90-negative. Rat MSCs have been reported to be CD71-negative. Our previous study also shows that rabbit MSCs are CD29-negative. However, human MSCs are generally considered to be CD29-, CD71-, and CD90-positive. Therefore, the surface markers of human MSCs might differ from those of other species. Rabbit bone marrow MSCs were obtained that had a multi-differentiation potential. The phenotype of these cells was studied using flow cytometry antibodies for 25 rabbit surface markers, namely, CD13, CD14, CD29, CD31, CD34, CD44, CD45, CD49d, CD49f, CD51, CD54, CD59, CD71, CD73, CD90, CD105, CD106, CD133, CD166, MHC I, MHC II, α-smooth muscle actin (α-SMA), cytokeratin, desmin, and vimentin. The phenotype of commercially available human MSCs was similarly studied using antibodies for human surface markers. CD14, CD31, CD34, CD45, CD49d, CD49f, CD51, CD54, CD71, CD106, CD133, MHC II, and cytokeratin were absent from both rabbit and human MSCs, while CD44, α-SMA, and vimentin were present on both cell lines. CD13, CD29, CD59, CD73, CD90, CD105, CD166, and MHC I were present on human MSCs, but not on rabbit MSCs. However, desmin was present on rabbit MSCs, but not on human MSCs. In total, the surface expression of nine markers differed between human and rabbit MSCs, whereas the surface expression of 16 markers was the same in the two cell lines.     

Differentiation of Human Embryonic Stem Cells into Cells with Corneal Keratocyte Phenotype

Corneal transparency depends on a unique extracellular matrix secreted by stromal keratocytes, mesenchymal cells of neural crest lineage. Derivation of keratocytes from human embryonic stem (hES) cells could elucidate the keratocyte developmental pathway and open a potential for cell-based therapy for corneal blindness. This study seeks to identify conditions inducing differentiation of pluripotent hES cells to the keratocyte lineage. Neural differentiation of hES cell line WA01(H1) was induced by co-culture with mouse PA6 fibroblasts. After 6 days of co-culture, hES cells expressing cell-surface NGFR protein (CD271, p75NTR) were isolated by immunoaffinity adsorption, and cultured as a monolayer for one week. Keratocyte phenotype was induced by substratum-independent pellet culture in serum-free medium containing ascorbate. Gene expression, examined by quantitative RT-PCR, found hES cells co-cultured with PA6 cells for 6 days to upregulate expression of neural crest genes including NGFR, SNAI1, NTRK3, SOX9, and MSX1. Isolated NGFR-expressing cells were free of PA6 feeder cells. After expansion as a monolayer, mRNAs typifying adult stromal stem cells were detected, including BMI1, KIT, NES, NOTCH1, and SIX2. When these cells were cultured as substratum-free pellets keratocyte markers AQP1, B3GNT7, PTDGS, and ALDH3A1 were upregulated. mRNA for keratocan (KERA), a cornea-specific proteoglycan, was upregulated more than 10,000 fold. Culture medium from pellets contained high molecular weight keratocan modified with keratan sulfate, a unique molecular component of corneal stroma. These results show hES cells can be induced to differentiate into keratocytes in vitro. Pluripotent stem cells, therefore, may provide a renewable source of material for development of treatment of corneal stromal opacities.     

The Generation of Six Clinical-Grade Human Embryonic Stem Cell Lines

The edict for producing clinically compliant human embryonic stem cells (hESCs) necessitates adherence to global ethical standards for egg procurement and embryo donation, conformity to regulations controlling clinical-grade cell and tissue product development, and compliance with current good tissue and manufacturing practices (cGTPs and cGMPs, respectively). For example, the U.S. FDA Center for Biologics Evaluation and Research recently promulgated regulations regarding human cells and cellular-based products (HCT/Ps) intended for tissue repair or replacement. Issued under Code of Federal Regulations parts 1270 and 1271 (Code of Federal Regulations, 2006a, 2006b), the rules are broadened by requirements for donor selection and cGMPs for HCT/Ps. By adhering to regulations and in anticipation of future standards, we have generated six clinical-grade hESC lines. Here we describe their manufacture, from embryo procurement to line characterization, including sterility and pathogen testing (Figure 1). To our knowledge, the lines represent the first to have been produced in compliance with international regulatory requirements, suitable for therapeutic use.     

STELLA Facilitates Differentiation of Germ Cell and Endodermal Lineages of Human Embryonic Stem Cells

Stella is a developmentally regulated gene highly expressed in mouse embryonic stem (ES) cells and in primordial germ cells (PGCs). In human, the gene encoding the STELLA homologue lies on chromosome 12p, which is frequently amplified in long-term cultured human ES cells. However, the role played by STELLA in human ES cells has not been reported. In the present study, we show that during retinoic acid (RA)-induced differentiation of human ES cells, expression of STELLA follows that of VASA, a marker of germline differentiation. By contrast, human embryonal carcinoma cells express STELLA at a higher level compared with both karyotypically normal and abnormal human ES cell lines. We found that over-expression of STELLA does not interfere with maintenance of the stem cell state of human ES cells, but following retinoic acid induction it leads to up-regulation of germline- and endodermal-associated genes, whereas neural markers PAX6 and NEUROD1 are down-regulated. Further, STELLA over-expression facilitates the differentiation of human ES cells into BE12-positive cells, in which the expression of germline- and endodermal-associated genes is enriched, and suppresses differentiation of the neural lineage. Taken together, this finding suggests a role for STELLA in facilitating germline and endodermal differentiation of human ES cells.     

Human Embryonic Stem Cells: Frontiers in Basic Research and Generation of Cell Technologies (Symposium and Advanced Training Course on Problems of Human Embryonic Stem Cells)

Russian Journal of Developmental Biology -     

人诱导多能干细胞与人胚胎干细胞分化过程中Oct4/Nanog基因表达的比较

目的：比较通过慢病毒方法获得的人诱导多能性干细胞（iPSCs）与人胚胎干细胞（hESCs）分化过程中全能性基因Oct4、Nanog的表达变化。方法：收集分化不同时间点的拟胚体（EBs）,检测三胚层分化基因以及全能性基因Oct4/Nanog的表达,并通过畸胎瘤组织切片的荧光染色分析Oct4的表达。结果：iPSCs获得的EB中内外三胚层分化基因表达的出现明显晚于hESCs来源的EB。不同于hESCs,iPSCs悬浮培养获得的EBs在体外培养18天未见内源性Oct4、Nanog基因表达的下调。未分化的iPSCs注射严重联合免疫缺陷（SCID）小鼠培养10周后获得的畸胎瘤中仍存在Oct4阳性的细胞,但iPS-#2中明显少于iPS-#5。结论：通过慢病毒方法获得的iPSCs虽然具有向三胚层分化的能力,但在分化过程中仍维持较高水平的全能性基因Oct4、Nanog的表达。     

The Development of Haematopoietic Cells Is Biased in Embryonic Stem Cell Chimaeras

The haematopoietic development of embryonic stem (ES) cell injection chimaeras was analysed using β-galactosidase expression from an X-linked transgene as a marker to distinguish the ES-derived cell population from the host cells. The number of cells in the different haematopoietic cell subpopulations was determined by flow cytometry. When the proportions of ES-derived cells in the antigen-positive lineages were compared to the ES cell contribution to all cells in the organs, we found an unexpected bias in the haematopoietic differentiation of ES-derived cells. ES descendants were overrepresented in the bone marrow B lymphoid cell population and the splenic myeloid cells but were underrepresented in the CD4-positive T lymphoid cells in the spleen. These results were obtained by comparison with control female animals that were X chromosome mosaic for β-galactosidase expression. These findings of uneven contribution to haematopoietic development by ES cells indicate that the commitment of ES cell descendants may be different from that of the host cells.     

Early Cell Fate Decisions of Human Embryonic Stem Cells and Mouse Epiblast Stem Cells Are Controlled by the Same Signalling Pathways

Human embryonic stem cells have unique value for regenerative medicine, as they are capable of differentiating into a broad variety of cell types. Therefore, defining the signalling pathways that control early cell fate decisions of pluripotent stem cells represents a major task. Moreover, modelling the early steps of embryonic development in vitro may provide the best approach to produce cell types with native properties. Here, we analysed the function of key developmental growth factors such as Activin, FGF and BMP in the control of early cell fate decisions of human pluripotent stem cells. This analysis resulted in the development and validation of chemically defined culture conditions for achieving specification of human embryonic stem cells into neuroectoderm, mesendoderm and into extra-embryonic tissues. Importantly, these defined culture conditions are devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Importantly, the growth factor roles defined using these culture conditions similarly drove differentiation of mouse epiblast stem cells derived from post implantation embryos, thereby reinforcing the hypothesis that epiblast stem cells share a common embryonic identity with human pluripotent stem cells. Therefore the defined growth factor conditions described here represent an essential step toward the production of mature cell types from pluripotent stem cells in conditions fully compatible with clinical use ant also provide a general approach for modelling the early steps of mammalian embryonic development.     

Sialyl-lactotetra,a Novel Cell Surface Marker of Undifferentiated Human Pluripotent Stem Cells

Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10⁹ cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells.     

Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.