Heart diseases are major causes of morbidity and mortality linked to extensive loss of cardiac cells. Embryonic stem cells (ESCs) give rise to cardiomyocyte-like cells, which may be used in heart cell replacement therapies. Most cardiogenic differentiation protocols involve the culture of ESCs as embryoid bodies (EBs). Stirred-suspension bioreactor cultures of ESC aggregates may be employed for scaling up the production of cardiomyocyte progeny but the wide range of EB sizes and the unknown effects of the hydrodynamic environment on differentiating EBs are some of the major challenges in tightly controlling the differentiation outcome. Here, we explored the cardiogenic potential of mouse ESCs (mESCs) and human ESCs (hESCs) encapsulated in poly-L-lysine (pLL)-coated alginate capsules. Liquefaction of the capsule core led to the formation of single ESC aggregates within each bead and their average size depended on the concentration of seeded ESCs. Encapsulated mESCs were directed along cardiomyogenic lineages in dishes under serum-free conditions with the addition of bone morphogenetic protein 4 (BMP4). Human ESCs in pLL-layered liquid core (LC) alginate beads were also differentiated towards heart cells in serum-containing media. Besides the robust cell proliferation, higher fractions of cells expressing cardiac markers were detected in ESCs encapsulated in LC than in solid beads. Furthermore, we demonstrated for the first time that ESCs encapsulated in pLL-layered LC alginate beads can be coaxed towards heart cells in stirred-suspension bioreactors. Encapsulated ESCs yielded higher fractions of Nkx2.5- and GATA4-positive cells in the biore-actor compared to dish cultures. Differentiated cells formed beating foci that responded to chronotropic agents in an organotypic manner. Our findings warrant further development and implementation of microencapsulation technologies in conjunction with bioreactor cultivation to enable the production of stem cell-derived cardiac cells appropriate for clinical therapies and applications.
Embryonic stem cells (ESCs); Heart cells; Directed differentiation; Encapsulation; Bioreactor
The use of embryonic stem cells (ESCs) for regenerative medicine has generated increased attention due to the favorable attributes of these cells; namely, they are pluripotent and possess long-term self-renewal capacity. The initial aims of the present study were: (i) to use stirred suspension bioreactors to expand and differentiate ESCs into osteogenic and chondrogenic cell types and (ii) to explore if these ESC-derived cells influenced skeletal healing in an in vivo fracture model. We show that differentiation protocols used in static culture are insufficient when applied directly to suspension culture bioreactors. Moreover, when bioreactor-differentiated cells are transplanted into a burr-hole defect in bone, severe disruption of the bone architecture was noted at the fracture site, as determined by microcomputed tomography (microCT) imaging and histopathology. Further characterization of the bioreactor-differentiated cultures revealed that a subpopulation of cells in the resulting aggregates expressed the pluripotency marker Oct-4 in the nucleus. Nuclear Oct-4 expression persisted even after 30 days of culture in the absence of leukemia inhibitory factor (LIF). Remarkably, and unlike ESCs differentiated into skeletal cell types in static cultures, bioreactor-differentiated aggregates implanted subcutaneously into SCID mice formed teratomas. The development of effective ESC differentiation protocols for suspension bioreactors will require a more complete understanding of the environmental conditions within these culture systems and the influence that these conditions have on the regulation of pluripotency and differentiation in ESCs.
Embryonic stem cells (ESCs) with their abilities for extensive proliferation and multi-lineage differentiation can serve as a renewable source of cellular material in regenerative medicine. However, the development of processes for large-scale generation of human ESCs (hESCs) or their progeny will be necessary before hESC-based therapies become a reality. We hypothesized that microcarrier stirred-suspension bioreactors characterized by scalability, straightforward operation, and tight control of the culture environment can be used for hESC culture and directed differentiation. Under appropriate conditions, the concentration of hESCs cultured in a microcarrier bioreactor increased 34- to 45-fold over 8 days. The cells retained the expression of pluripotency markers such as OCT3/4A, NANOG, and SSEA4, as assessed by quantitative PCR, immunocytochemistry, and flow cytometry. We further hypothesized that hESCs on microcarriers can be induced to definitive endoderm (DE) when incubated with physiologically relevant factors. In contrast to embryoid body cultures, all hESCs on microcarriers are exposed to soluble stimuli in the bulk medium facilitating efficient transition to DE. After reaching a peak concentration, hESCs in microcarrier cultures were incubated in medium containing activin A, Wnt3a, and low concentration of serum. More than 80% of differentiated hESCs coexpressed FOXA2 and SOX17 in addition to other DE markers, whereas the expression of non-DE genes was either absent or minimal. We also demonstrate that the hESC-to-DE induction in microcarrier cultures is scalable. Our findings support the use of microcarrier bioreactors for the generation of endoderm progeny from hESCs including pancreatic islets and liver cells in therapeutically useful quantities.
Advances in stem cell biology have afforded promising results for the generation of various cell types for therapies against devastating diseases. However, a prerequisite for realizing the therapeutic potential of stem cells is the development of bioprocesses for the production of stem cell progeny in quantities that satisfy clinical demands. Recent reports on the expansion and directed differentiation of human embryonic stem cells (hESCs) in scalable stirred-suspension bioreactors (SSBs) demonstrated that large-scale production of therapeutically useful hESC progeny is feasible with current state-of-the-art culture technologies. Stem cells have been cultured in SSBs as aggregates, in microcarrier suspension and after encapsulation. The various modes in which SSBs can be employed for the cultivation of hESCs and human induced pluripotent stem cells (hiPSCs) are described. To that end, this is the first account of hiPSC cultivation in a microcarrier stirred-suspension system. Given that cultured stem cells and their differentiated progeny are the actual products used in tissue engineering and cell therapies, the impact of bioreactor's operating conditions on stem cell self-renewal and commitment should be considered. The effects of variables specific to SSB operation on stem cell physiology are discussed. Finally, major challenges are presented which remain to be addressed before the mainstream use of SSBs for the large-scale culture of hESCs and hiPSCs.
Mouse embryonic stem cell (ESC) lines, and more recently human ESC lines, have become valuable tools for studying early mammalian development. Increasing interest in ESCs and their differentiated progeny in drug discovery and as potential therapeutic agents has highlighted the fact that current two-dimensional (2D) static culturing techniques are inadequate for large-scale production. The culture of mammalian cells in three-dimensional (3D) agitated systems has been shown to overcome many of the restrictions of 2D and is therefore likely to be effective for ESC proliferation. Using murine ESCs as our initial model, we investigated the effectiveness of different 3D culture environments for the expansion of pluripotent ESCs. Solohill Collagen, Solohill FACT, and Cultispher-S microcarriers were employed and used in conjunction with stirred bioreactors. Initial seeding parameters, including cell number and agitation conditions, were found to be critical in promoting attachment to microcarriers and minimizing the size of aggregates formed. While all microcarriers supported the growth of undifferentiated mESCs, Cultispher-S out-performed the Solohill microcarriers. When cultured for successive passages on Cultispher-S microcarriers, mESCs maintained their pluripotency, demonstrated by self-renewal, expression of pluripotency markers and the ability to undergo multi-lineage differentiation. When these optimized conditions were applied to unweaned human ESCs, Cultispher-S microcarriers supported the growth of hESCs that retained expression of pluripotency markers including SSEA4, Tra-1–60, NANOG, and OCT-4. Our study highlights the importance of optimization of initial seeding parameters and provides proof-of-concept data demonstrating the utility of microcarriers and bioreactors for the expansion of hESCs. Biotechnol. Bioeng. 2010;107:683–695. © 2010 Wiley Periodicals, Inc.
embryonic stem cells; 3D agitated suspension culture; microcarriers; pluripotency; bioreactors
Human embryonic stem cells (hESCs) can proliferate for a prolonged period and differentiate into cardiomyocytes in vitro. Recent studies used bone morphogenetic protein 2 (BMP2) to generate cardiomyocytes from hESCs, however, all those studies used early embryoid bodies (EBs) and did not retrieve cardiomyocytes with a high yield. In this study, we treated long-term cultured EBs with BMP2 in order to promote differentiation into cardiomyocytes from hESCs.
Materials and Methods
hESC lines, including SNUhES3 and SNUhES4, were used in this study. Undifferentiated hESC colonies were detached to form EBs and cultured for up to 30 days. These long-term cultured EBs were differentiated into cardiomyocytes in serum-containing media. In our protocol, BMP2 was applied for 5 days after attachment of EBs. Cardiac specific markers, beating of differentiated cells and electron microscopic (EM) ultrastructures were evaluated and analyzed.
Compared to 10-day or 20-day EBs, 30-day EBs showed a higher expression level of cardiac specific markers, Nkx2.5 and α-myosin heavy chain (αMHC). Treatment of BMP2 increased expression of cardiac troponin (cTn) I and α-actinin when evaluated at 20 days after attachment of 30-day EBs. Beating of differentiated cells was observed from 7 to 20 days after attachment. Moreover, EM findings demonstrated fine structures such as Z bands in these differentiated cardiomyocytes. These long-term cultured EBs yielded cardiomyocytes with an efficiency of as high as 73.6% when assessed by FACS.
We demonstrated that the use
of long-term cultured EBs may enhance differentiation into cardiomyocytes from hESCs when treated with BMP2.
Bone morphogenetic protein 2; cardiomyocytes; cell differentiation; embryoid bodies; embryonic stem cells; long-term
Due to their self-renewal, embryonic stem cells (ESCs) are attractive cells for applications in regenerative medicine and tissue engineering. Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best. It is possible that the traditional culture methods, which have been used, are not optimal and that other approaches must be explored.
ESCs were differentiated into osteoblast lineage using a micro-mass approach. In response to osteogenic differentiation medium, many cells underwent apoptosis, while others left the micro-mass, forming small aggregates in suspension. These aggregates were cultured in three different culture conditions (adhesion, static suspension, and stirred suspension), then examined for osteogenic potential in vitro and in vivo. In adhesion culture, ESCs primed to become osteoblasts recommitted to the adipocyte lineage in vitro. In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification. In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells.
We demonstrate that microenvironment modulates cell fate and subsequent tissue formation during ESC differentiation. For effective tissue engineering using ESCs, it is important to develop optimized cell culture/differentiation conditions based upon the influence of microenvironment.
The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) aggregates and iii) immobilized on microcarriers. In order to establish a scalable bioprocess, hESC-microcapsules were cultured in stirred tank bioreactors.
The combination of microencapsulation and microcarrier technology resulted in a highly efficient protocol for the production and storage of pluripotent hESCs. This strategy ensured high expansion ratios (an approximately twenty-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation. When compared with non-encapsulated cells, cell survival post-thawing demonstrated a three-fold improvement without compromising hESC characteristics.
Microencapsulation also improved the culture of hESC aggregates by protecting cells from hydrodynamic shear stress, controlling aggregate size and maintaining cell pluripotency for two weeks.
This work establishes that microencapsulation technology may prove a powerful tool for integrating the expansion and cryopreservation of pluripotent hESCs. The 3D culture strategy developed herein represents a significant breakthrough towards the implementation of hESCs in clinical and industrial applications.
Debilitating cardiomyocyte loss underlies the progression to heart failure. Although there have been significant advances in treatment, current therapies are intended to improve or preserve heart function rather than regenerate lost myocardium. A major hurdle in implementing a cell-based regenerative therapy is the inefficient differentiation of cardiomyocytes from either endogenous or exogenous stem cell sources. Moreover, cardiomyocytes that develop in human embryonic stem cell (hESC) or human-induced pluripotent stem cell (hIPSC) cultures are comparatively immature, even after prolonged culture, and differences in their calcium handling, ion channel, and force generation properties relative to adult cardiomyocytes raise concerns of improper integration and function after transplantation. Thus, the discovery of natural and novel small molecule synthetic regulators of differentiation and maturation would accelerate the development of stem-cell-based myocardial therapies. Here, we document recent advances in defining natural signaling pathways that direct the multistep cardiomyogenic differentiation program and the development of small molecules that might be used to enhance differentiation as well as the potential characteristics of lead candidates for pharmaceutical stimulation of endogenous myocardial replacement.
Cardiomyocyte; Embryonic stem cell; Chemical screening
Therapeutic and industrial applications of pluripotent stem cells and their derivatives require large cell quantities generated in defined conditions. To this end, we have translated single cell-inoculated suspension cultures of human pluripotent stem cells (hPSCs; including human induced pluripotent stem cells [hiPS] and human embryonic stem cells [hESC]) to stirred tank bioreactors. These systems that are widely used in biopharmaceutical industry allow straightforward scale up and detailed online monitoring of key process parameters. To ensure minimum medium consumption, but in parallel functional integration of all probes mandatory for process monitoring, that is, for pO2 and pH, experiments were performed in 100 mL culture volume in a “mini reactor platform” consisting of four independently controlled vessels. By establishing defined parameters for tightly controlled cell inoculation and aggregate formation up to 2×108 hiPSCs/100 mL were generated in a single process run in 7 days. Expression of pluripotency markers and ability of cells to differentiate into derivates of all three germ layers in vitro was maintained, underlining practical utility of this new process. The presented data provide key steps toward scalable mass expansion of human iPS and ES cells thereby enabling translation of stem cell research to (pre)clinical application in relevant large animal models and valuable in vitro assays for drug development and validation as well.
Proteoglycans and hyaluronan play critical roles in heart development. In this study, human embryonic stem cells (hESC) were used as a model to quantify the synthesis of proteoglycans and hyaluronan in hESC in the early stages of differentiation, and after directed differentiation into cardiomyocytes. We demonstrated that both hESC and cardiomyocyte cultures synthesize an extracellular matrix (ECM) enriched in proteoglycans and hyaluronan. During cardiomyocyte differentiation, total proteoglycan and hyaluronan decreased and the proportion of proteoglycans bearing heparan sulfate chains was reduced. Versican, a chondroitin sulfate proteoglycan, accumulated in hESC and cardiomyocyte cultures. Furthermore, versican synthesized by hESC contained more N- and O-linked oligosaccharide than versican from cardiomyocytes. Transcripts for the versican variants, V0, V1, V2 and V3, increased in cardiomyocytes compared to hESC, with V1 most abundant. Hyaluronan in hESC had lower molecular weight than hyaluronan from cardiomyocyte cultures. These changes were accompanied by an increase in HAS-1 and HAS-2 mRNA in cardiomyocyte cultures, with HAS-2 most abundant. Interestingly, HAS-3 was absent from the cardiomyocyte cultures, but expressed by hESC. These results indicate that human cardiomyocyte differentiation is accompanied by specific changes in the expression and accumulation of ECM components and suggest a role for versican and hyaluronan in this process.
versican; hyaluronan; cardiomyocyte; differentiation; stem cells; glycosylation
Embryonic stem cell (ESC)–derived cardiomyocytes are anticipated to serve as a useful source for future cell-based cardiovascular disease therapies. Research emphasis is currently focused on determining methods to direct the differentiation of ESCs to a large population of cardiomyocytes with high purity. To this aim, understanding the molecular mechanisms that control ESC-to-cardiomyocyte differentiation should play a critical role in the development of this methodology. The Wnt/β-catenin signaling pathway has been implicated in both embryonic cardiac development and in vitro ESC differentiation into cardiomyocytes. Chibby is a recently identified nuclear protein that directly binds to β-catenin and antagonizes its transcriptional activity.
Methods and Results
Chibby was ubiquitously expressed in early stages of ESC differentiation but upregulated during cardiomyocyte specification. Of interest, the Chibby gene promoter has multiple binding sites for the cardiac-specific homeodomain protein Nkx2.5, and its promoter activity was indeed positively regulated by Nkx2.5. Furthermore, overexpression of Chibby increased cardiac differentiation of ESCs, whereas loss of Chibby by RNAi impaired cardiomyocyte differentiation.
These data illustrate the regulation and function of Chibby in facilitating cardiomyocyte differentiation from ESCs. By revealing molecular mechanisms that control ESC-to-cardiomyocyte differentiation, this study will allow for the future development of technologies to improve cardiomyocyte differentiation from ESCs.
myocytes; signal transduction; stem cells
Human embryonic stem cells (hESCs) have the potential to provide an unlimited source of cardiomyocytes, which are invaluable resources for drug or toxicology screening, medical research, and cell therapy. Currently a number of obstacles exist such as the insufficient efficiency of differentiation protocols, which should be overcome before hESC-derived cardiomyocytes can be used for clinical applications. Although the differentiation efficiency can be improved by the genetic manipulation of hESCs to over-express cardiac-specific transcription factors, these differentiated cells are not safe enough to be applied in cell therapy. Protein transduction has been demonstrated as an alternative approach for increasing the efficiency of hESCs differentiation toward cardiomyocytes.
We present an efficient protocol for the differentiation of hESCs in suspension by direct introduction of a LIM homeodomain transcription factor, Islet1 (ISL1) recombinant protein into the cells.
We found that the highest beating clusters were derived by continuous treatment of hESCs with 40 µg/ml recombinant ISL1 protein during days 1–8 after the initiation of differentiation. The treatment resulted in up to a 3-fold increase in the number of beating areas. In addition, the number of cells that expressed cardiac specific markers (cTnT, CONNEXIN 43, ACTININ, and GATA4) doubled. This protocol was also reproducible for another hESC line.
This study has presented a new, efficient, and reproducible procedure for cardiomyocytes differentiation. Our results will pave the way for scaled up and controlled differentiation of hESCs to be used for biomedical applications in a bioreactor culture system.
High proliferative and differentiation capacity renders embryonic stem cells (ESCs) a promising cell source for tissue engineering and cell-based therapies. Harnessing their potential, however, requires well-designed, efficient and reproducible expansion and differentiation protocols as well as avoiding hazardous by-products, such as teratoma formation. Traditional, standard culture methodologies are fragmented and limited in their fed-batch feeding strategies that afford a sub-optimal environment for cellular metabolism. Herein, we investigate the impact of metabolic stress as a result of inefficient feeding utilizing a novel perfusion bioreactor and a mathematical model to achieve bioprocess improvement.
To characterize nutritional requirements, the expansion of undifferentiated murine ESCs (mESCs) encapsulated in hydrogels was performed in batch and perfusion cultures using bioreactors. Despite sufficient nutrient and growth factor provision, the accumulation of inhibitory metabolites resulted in the unscheduled differentiation of mESCs and a decline in their cell numbers in the batch cultures. In contrast, perfusion cultures maintained metabolite concentration below toxic levels, resulting in the robust expansion (>16-fold) of high quality ‘naïve’ mESCs within 4 days. A multi-scale mathematical model describing population segregated growth kinetics, metabolism and the expression of selected pluripotency (‘stemness’) genes was implemented to maximize information from available experimental data. A global sensitivity analysis (GSA) was employed that identified significant (6/29) model parameters and enabled model validation. Predicting the preferential propagation of undifferentiated ESCs in perfusion culture conditions demonstrates synchrony between theory and experiment.
The limitations of batch culture highlight the importance of cellular metabolism in maintaining pluripotency, which necessitates the design of suitable ESC bioprocesses. We propose a novel investigational framework that integrates a novel perfusion culture platform (controlled metabolic conditions) with mathematical modeling (information maximization) to enhance ESC bioprocess productivity and facilitate bioprocess optimization.
Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1+ common progenitor cells, and identified highly cardiogenic progenitors as Flk1+/CXCR4+/VE-cadherin− (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1+ cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1+ cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.
Differentiation of embryonic stem cell (ESC)-derived embryoid bodies (EBs) is a heterogeneous process. ESCs can differentiate in vitro into different cell types including beating cardiomyocytes. The main aim of the present study was to develop an improved preparation method for scanning electron microscopic study of ESC-derived cardiac bundles and to investigate the fine structural characteristics of mouse ESCs-derived cardiomyocytes using electron microscopy. The mouse ESCs differentiation was induced by EBs’ development through hanging drop, suspension and plating stages. Cardiomyocytes appeared in the EBs’ outgrowth as beating clusters that grew in size and formed thick branching bundles gradually. Cardiac bundles showed cross striation even when they were observed under an inverted microscope. They showed a positive immunostaining for cardiac troponin I and α-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESC-derived cardiomyocytes. Three weeks after plating, differentiated EBs showed a superficial layer of compact fibrous ECM that made detailed observation of cardiac bundles impossible. We tried several preparation methods to remove unwanted cells and fibers, and finally we revealed the branching bundles of cardiomyocytes. In TEM study, most cardiomyocytes showed parallel arrays of myofibrils with a mature sarcomeric organization marked by H-bands, M-lines and numerous T-tubules. Cardiomyocytes were connected to each other by intercalated discs composed of numerous gap junctions and fascia adherences.
Electronic supplementary material
The online version of this article (doi:10.1007/s10616-011-9411-4) contains supplementary material, which is available to authorized users.
Mouse embryonic stem cell; Cardiomyocyte; Scanning electron microscopy; Ultrastructure
Pluripotent Human Embryonic Stem Cells (hESCs) have the unconstrained capacity for long-term stable undifferentiated growth in culture and unrestricted developmental capacity. Packaging of the eukaryotic genome into chromatin confers higher order structural control over maintaining stem cell plasticity and directing differentiation. We recently reported the establishment of a defined culture system for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards functional lineages. To unveil the epigenetic mechanism in maintaining the epiblast pluripotence of hESCs, in this study, the global chromatin dynamics in the pluripotent hESCs maintained under the defined culture were examined. This study shows that the genomic plasticity of pluripotent hESCs is enabled by an acetylated globally active chromatin maintained by Oct-4. The pluripotency of hESCs that display normal stable expansion is associated with high levels of expression and nuclear localization of active chromatin remodeling factors that include acetylated histone H3 and H4, Brg-1, hSNF2H, HAT p300, and HDAC1; weak expression or cytoplasmic localization of repressive chromatin remodeling factors that are implicated in transcriptional silencing; and residual H3 K9 methylation. A dynamic progression from acetylated to transient hyperacetylated to hypoacetylated chromatin states correlates with loss-of-Oct4-associated hESC differentiation. RNA interference directed against Oct-4 and HDAC inhibitor analysis support this pivotal link between chromatin dynamics and hESC differentiation. These findings reveal an epigenetic mechanism for placing global chromatin dynamics as central to tracking the normal pluripotency and lineage progression of hESCs.
Human embryonic stem cells; Pluripotency; Epigenetic; Chromatin; Histone modification; Histone acetylation; Histone deacetylation; Histone methylation; Histone acetyltransferase; Histone deacetylase; Histone methytransferase; Chromatin remodeling; Oct-4; Differentiation; Defined culture system
Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs) are promising cell sources for cardiac regenerative medicine. To realize hESC/hiPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes are required. Though marker gene transduction or fluorescent-based purification methods have been reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purpose but have not yet been established. In this study, we attempted to identify cell surface markers for cardiomyocytes derived from hESC/hiPSCs.
Method and Result
We adopted a previously reported differentiation protocol for hESCs based on high density monolayer culture to hiPSCs with some modification. Cardiac troponin-T (TNNT2)-positive cardiomyocytes appeared robustly with 30–70% efficiency. Using this differentiation method, we screened 242 antibodies for human cell surface molecules to isolate cardiomyocytes derived from hiPSCs and identified anti-VCAM1 (Vascular cell adhesion molecule 1) antibody specifically marked cardiomyocytes. TNNT2-positive cells were detected at day 7–8 after induction and 80% of them became VCAM1-positive by day 11. Approximately 95–98% of VCAM1-positive cells at day 11 were positive for TNNT2. VCAM1 was exclusive with CD144 (endothelium), CD140b (pericytes) and TRA-1-60 (undifferentiated hESCs/hiPSCs). 95% of MACS-purified cells were positive for TNNT2. MACS purification yielded 5−10×105 VCAM1-positive cells from a single well of a six-well culture plate. Purified VCAM1-positive cells displayed molecular and functional features of cardiomyocytes. VCAM1 also specifically marked cardiomyocytes derived from other hESC or hiPSC lines.
We succeeded in efficiently inducing cardiomyocytes from hESCs/hiPSCs and identifying VCAM1 as a potent cell surface marker for robust, efficient and scalable purification of cardiomyocytes from hESC/hiPSCs. These findings would offer a valuable technological basis for hESC/hiPSC-based cell therapy.
Embryonic stem cells (ESCs) are pluripotent cells capable of differentiating into all somatic and germ cell types. The intrinsic ability of pluripotent cells to generate a vast array of different cells makes ESCs a robust resource for a variety of cell transplantation and tissue engineering applications, however, efficient and controlled means of directing ESC differentiation is essential for the development of regenerative therapies. ESCs are commonly differentiated in vitro by spontaneously self-assembling in suspension culture into 3D cell aggregates called embryoid bodies (EBs), which mimic many of the hallmarks of early embryonic development, yet the 3D organization and structure of EBs also presents unique challenges to effectively direct the differentiation of the cells. ESC differentiation is strongly influenced by physical and chemical signals comprising the local extracellular microenvironment, thus current methods to engineer EB differentiation have focused primarily on spatially controlling EB size, adding soluble factors to the media, or culturing EBs on or within natural or synthetic extracellular matrices. While most such strategies aim to influence differentiation from the exterior of EBs, engineering the microenvironment directly within EBs enables new opportunities to efficiently direct the fate of the cells by locally controlling the presentation of morphogenic cues.
embryoid body; embryonic stem cells; differentiation; microenvironment; morphogenesis
Pluripotent stem cells represent one promising source for cellular cardiomyoplasty. In this study, we employed cardiac magnetic resonance (CMR) to examine the ability of highly enriched cardiomyocytes (CMs) derived from murine embryonic stem cells (ESC) to form grafts and improve contractile function of infarcted rat hearts.
Methods and Results
Highly enriched ESC-CMs were obtained by inducing cardiac differentiation of ESCs stably expressing a cardiac restricted puromycin resistance gene. At the time of transplantation, enriched ESC-CMs expressed cardiac specific markers and markers of developing CMs but only 6% of them were proliferating. A growth factor containing vehicle solution or ESC-CMs (5–10 million) suspended in the same solution was injected into athymic rat hearts one week after myocardial infarction (MI). Initial infarct size was measured by CMR one day post-MI. Compared to vehicle, treatment with ESC-CMs improved global systolic function at 1 and 2-months post injection, and significantly increased contractile function in initially infarcted and border zones. Immunohistochemistry confirmed successful engraftment and the persistence of α-actinin positive ESC-CMs that also expressed α-smooth muscle actin. Connexin-43 positive sites were observed between grafted ESC-CMs but only rarely between grafted and host CMs. No teratomas were observed in any of the animals.
Highly enriched and early staged ESC-CMs were safe, formed stable grafts and mediated a long-term recovery of global and regional myocardial contractile function following infarction.
cardiac magnetic resonance imaging; embryonic stem cells; left ventricular remodeling; left ventricular wall motion; myocardial infarction
Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca2+ signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca2+ signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca2+ handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques.
Cardiomyocytes derived from pluripotent stem cells can be applied in drug testing, disease modeling and cell-based therapy. However, without procardiogenic growth factors, the efficiency of cardiomyogenesis from pluripotent stem cells is usually low and the resulting cardiomyocyte population is heterogeneous. Here, we demonstrate that induced pluripotent stem cells (iPSCs) can be derived from murine ventricular myocytes (VMs), and consistent with other reports of iPSCs derived from various somatic cell types, VM-derived iPSCs (ViPSCs) exhibit a markedly higher propensity to spontaneously differentiate into beating cardiomyocytes as compared to genetically matched embryonic stem cells (ESCs) or iPSCs derived from tail-tip fibroblasts. Strikingly, the majority of ViPSC-derived cardiomyocytes display a ventricular phenotype. The enhanced ventricular myogenesis in ViPSCs is mediated via increased numbers of cardiovascular progenitors at early stages of differentiation. In order to investigate the mechanism of enhanced ventricular myogenesis from ViPSCs, we performed global gene expression and DNA methylation analysis, which revealed a distinct epigenetic signature that may be involved in specifying the VM fate in pluripotent stem cells.
induced pluripotent stem cells; ventricular cardiomyocyte; regenerative medicine; epigenetic memory
Because pluripotent embryonic stem cells (ESCs) are able to differentiate into any tissue, they are attractive agents for tissue regeneration. Although improvement of cardiac function has been observed after transplantation of pluripotent ESCs, the extent to which these effects reflect ESC-mediated re-muscularization, re-vascularization or paracrine mechanisms is unknown. Moreover, because ESCs may generate teratomas, the ability to predict the outcome of cellular differentiation, especially when transplanting pluripotent ESCs, is essential; conversely, a requirement to use pre-differentiated ESCs would limit their application to highly characterized subsets that are available in limited numbers. In the experiments reported here we transplanted low numbers of two murine ESC lines, respectively engineered to express a β-galactosidase gene from either a constitutive (elongation factor, EF) or a cardiac-specific (α-myosin heavy chain, α-MHC) promoter, into infarcted mouse myocardium. Although ESC-derived tumors formed within the pericardial space in 21% of injected hearts, lacZ histochemistry revealed that engraftment of ESC was restricted to the ischemic myocardium. Echocardiographic monitoring of ESC-injected hearts that did not form tumors revealed functional improvements by four weeks post-infarction, including significant increases in ejection fraction (EF), circumferential fiber shortening velocity (VCF) and peak mitral bloodflow velocity (peak-E). These experiments indicate that the infarcted myocardial environment can support engraftment and cardiomyogenic differentiation of pluripotent ESCs, concomitant with partial functional recovery.
embryonic stem cells; echocardiography; elongation factor promoter; myosin heavy chain promoter; lacZ histochemistry; mouse myocardial infarction
Human embryonic stem cell (hESC) culture is routinely performed using inactivated mouse embryonic fibroblasts (MEFs) as a feeder cell layer (FL). Although these cells maintain pluripotency of hESCs, the molecular basis for this is unknown. Objectives of this study were to determine whether timing between MEF inactivation and their use as a FL influenced hESC growth and differentiation, and to begin defining the mechanism(s) involved. hESCs were plated on MEFs prepared 1 (MEF-1), 4 (MEF-4), and 7 (MEF-7) days earlier. hESC colony morphology and Oct3/4 expression levels were evaluated to determine the influence of different FLs. Significant enhancement of hESC growth (self-renewal) was observed on MEF-1 compared with MEF-4 and/or MEF-7. Conditioned media (CM) collected from MEF-1 supported significantly better hESC growth in a FL-free system compared to MEF-7 CM. Effects of MEFs on hESC growth were not caused by differences in cell density or viability, although indications of apoptosis were observed in MEF-7. Scanning electron microscopy demonstrated that MEF-7 were morphologically distinct from MEF-1 and MEF-4. Microarray analysis identified 19 genes related to apoptosis with significantly different levels of expression between MEF-1 and MEF-7. Several differentially expressed RNAs had gene ontology classifications associated with extracellular matrix (ECM) structural constituents and growth factors. Because members of Wnt signaling pathway were identified in the array analysis, we examined the ability of the Wnt1 CM and secreted frizzled-related proteins to affect hESC growth and differentiation. The addition of Wnt1 CM to both MEF-1 and MEF-7 significantly increased the number of undifferentiated colonies, while the addition of Sfrps promoted differentiation. Together, these results suggest that microenvironment, ECM, and soluble factors expressed by MEF-1 are significantly better at maintaining self-renewal and pluripotency of hESCs. Our findings have important implications in the optimization of hESC culture when MEFs are used as FL or CM is used in FL-free culture.
Embryonic Stem Cells (ESCs) can differentiate into cardiomyocytes (CMs) in vitro but the differentiation level from ESCs is low. Here we describe a simple co-culture model by commercially available Millicell™ hanging cell culture inserts to control the long-term differentiation of ESCs into CMs.
Mouse ESCs were cultured in hanging drops to form embryoid bodies (EBs) and treated with 0.1 mmol/L ascorbic acid to induce the differentiation of ESCs into CMs. In the indirect co-culture system, EBs were co-cultured with epidermal keratinocytes (EKs) or neonatal CMs (NCMs) by the hanging cell culture inserts (PET membranes with 1 µm pores). The molecular expressions and functional properties of ESC-derived CMs in prolonged culture course were evaluated. During time course of ESC differentiation, the percentages of EBs with contracting areas in NCMs co-culture were significantly higher than that without co-culture or in EKs co-culture. The functional maintenance of ESC-derived CMs were more prominent in NCMs co-culture model.
These results indicate that NCMs co-culture promote ESC differentiation and has a further effect on cell growth and differentiation. We assume that the improvement of the differentiating efficiency of ESCs into CMs in the co-culture system do not result from the effect of co-culture directly on cell differentiation, but rather by signaling effects that influence the cells in proliferation and long-term function maintenance.