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Directed differentiation of human mesenchymal stem cells toward a cardiomyogenic fate commitment through formation of cell aggregates
Biochemical Engineering Journal 84 (2014) 53–58
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular Article
Directed differentiation of human mesenchymal stem cells toward a cardiomyogenic fate commitment through formation of cell aggregates夽 Mee-Hae Kim a , Yuuki Ogawa a , Koji Yamada b , Masahito Taya b , Masahiro Kino-oka a,∗ a b
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan
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Article history: Received 16 August 2013 Received in revised form 9 November 2013 Accepted 23 December 2013 Available online 15 January 2014 Keywords: Animal cell culture Growth kinetics Tissue cell culture Biomedical Human mesenchymal stem cells Cardiomyogenic fate Dendrimer surface
a b s t r a c t Cell morphology is known to modulate the multipotential lineage commitment of stem cells. We provide a new strategy to induce the early lineage commitment of human mesenchymal stem cells (hMSCs) toward a cardiomyogenic fate through the formation of cell aggregates. A surface-immobilized polyamidoamine dendrimer with fifth generation of dendron structure was used during the culturing of hMSCs. These hMSCs cultured on the G5 surface formed aggregates through active migration and division. More than 22% of cardiac troponin-T (cTnT)-positive (cTnT+ ) cells in aggregates formed on the dendrimer surface; the population formed on the dendrimer surface was higher than that in conventional culture vessel. When cell aggregate was reseeded onto a fresh G5 surface, single cells migrated out of the aggregates, proliferated, and formed new aggregates. This passage method, accompanied with repetitive aggregate dispersion and formation, was applied to cultures over 40 days. The proportion of cTnT+ cells increased to 62% by the end of third passage. Our results suggest that culturing hMSCs on G5 surface results in directed commitment of the hMSCs toward a cardiomyocyte-like fate. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Mesenchymal stem cells (MSCs) are attracting increased attention because they possess multi-lineage developmental potential and the capacity for self-renewal in vivo [1,2]. MSCs are multipotent cells, initially isolated from the bone marrow, and are noted for their ability to differentiate into osteoblasts, chondrocytes, and adipocytes [2]. As an autologous source of stem cells, MSCs have been considered a possible source of cells that can be applied to regenerative therapies [1]. To facilitate in vitro development, many researchers have attempted to design microenvironments that mimic the stem cell niche, thereby driving cells down their preferred differentiation pathway [3–5]. The niche paradigm outlines three interactions involving cells, culture medium components, adjacent cells and substrates; these are cell-soluble factor, cell–cell, and cell–substrate interactions [3]. In vitro strategies for regulating stem cell behavior attempt to mimic developmental mechanisms and establish artificial environments that promote specific cell fates [2,5]. The majority of previous research studies have focused on dynamic process that are tightly orchestrated by the sequential
夽 Much of this work forms the basis of the Ph.D. dissertation of Yuuki Ogawa. ∗ Corresponding author. Tel.: +81 06 6879 7444; fax: +81 06 6879 4246. E-mail address: [email protected] (M. Kino-oka). 1369-703X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.12.015
expression of multiple signal transduction proteins and transcription factors working in a combination [4,6]. A number of signaling pathways (Wnts/Nodal) and growth factors (bone morphogenic proteins and fibroblast growth factor) have been implicated in the development of specialized cardiac subtypes [4–6]. The times and concentrations at which these factors affect differentiation toward the desired fate have been optimized. Although human MSCs (hMSCs) possess a self-renewing capacity, they display heterogeneous responses upon the induction of differentiation, resulting in a mixture of differentiated cells [5,6]. Zhang et al. [7] reported that culturing MSCs for 21 days with 5-azacytidine in culture medium induced 45% cardiomyocyte-like cells that were cardiac troponin-T positive (cTnT+ ); however, other differentiation pathways were also followed leading to a heterogeneous population of differentiated cells. Fully functional cardiomyocytes with striated cytoskeleton and proper electrical coupling for hMSCs have not been observed. Overall, it appears that the best differentiation strategies produce homogenous populations of cardiomyocytes; these strategies tend to be reproducible, result in cells of the appropriate quality, and produce large quantities of cells. In previous studies, the use of a dendrimer surface has been proposed as a method of regulating the morphology and function of cells [8–11]. Polyamidoamine dendrimers have been immobilized to culture surfaces, with the variations in generation number of the dendrimer surface shown to cause morphological changes in
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hMSCs. The observed morphological changes were accompanied by dynamic cytoskeletal formation in cells on the culture surface. Subsequent formation of cell aggregates led to cardiomyogenic differentiation of hMSCs in vitro [10]. It is believed that endogenous Rho family GTPase signaling pathways, accompanied by morphological changes, are an intrinsic cue for modulating transcription factors. In the present study, we used a fifth generation of dendrimer surface in an attempt to induce initiation of lineage commitment of hMSCs toward a cardiomyogenic fate. Novel approaches to cell culture including aggregate dispersion and formation during passage were trialed, and prolonged culturing was conducted to enhance the homogeneity of the cardiomyocyte-like cell population.
an antibody against cTnT (Materials and Methods in the Supporting Information). For quantitative assessment of cardiomyogenic differentiation, suspended cells were re-seeded after harvesting from the cultures as described above. The numbers of cTnT+ and the DAPI+ nuclei were evaluated to determine the ratio of cTnT+ (XP ) to DAPI+ (XT ) cells.
2. Materials and methods
3. Results
2.1. Cells and culture conditions
3.1. Dynamic behavior and spontaneous cardiomyogenic differentiation of hMSCs
Human bone marrow-derived MSCs were obtained from Lonza (Lot no. 8F3543; Walkersville, MD, USA). Routine subcultures of hMSCs were conducted in a 75-cm2 flask (Corning Costar, Cambridge, MA, USA) using hMSC growth medium (Lonza) at 37 ◦ C in a humidified atmosphere containing 5% CO2 . Upon reaching 70% confluence, cells were detached by enzymatic treatment with a 0.1% trypsin/0.02% EDTA solution (Sigma–Aldrich, St. Louis, MO, USA). Cells that had undergone less than five passages were used in subsequent experiments. For all experiments, hMSCs were expanded for the specified number of days in Dulbecco’s modified Eagle’s medium (DMEM; Sigma–Aldrich) supplemented with 10% fetal bovine serum (Invitrogen, Grand Island, NY, USA) and antibiotics (100 U/cm3 penicillin G, 0.1 mg/cm3 streptomycin and 0.25 mg/cm3 amphotericin B; Invitrogen). Cultures were grown in square 8-well plate with and without dendrimer immobilized to the surface. The seeding density was fixed at a viable cell concentration of 5.0 × 103 cells/cm2 (X0 ). Culture medium was replenished every 3 days. If necessary, round-bottomed 96-well plates were used to generate floating cell aggregates, which were used to prepare aggregates of hMSCs as controls. 2.2. Surface preparation The dendrimer surface was prepared using the conventional tissue culture polystyrene (PS) surface of a square 8-well plate (surface area; 10.5 cm2 , Nunc, Roskilde, Denmark), as described previous report [8]. Briefly, a G5 surface was created, under sterile conditions, by changing the generation number of synthesized dendrimers over four reactions. Hydroxyl groups were displayed on the plain surface by pouring potassium tert-butoxide into the wells. Then, aqueous glutaraldehyde was introduced into the wells, with the wells were then treated with a tris(2-aminoethyl) amine solution to produce a dendron structure. The wells were rinsed with sterile water and the previous step repeated until the fifth generation of dendrimers was synthesized. To display glucose as a terminal ligand, d-glucose was applied.
2.4. Statistical analysis All experiments were performed at least three times and data expressed as means with standard deviations. Student’s t-test was used to determine statistical significances among the data sets; pvalues less than 0.01 and 0.05 were considered significant.
Cultures of hMSCs were grown on G5 and PS surfaces in 8-well plates, and in round-bottom 96-well plates, with non-adhesive culture surfaces, for 10 days. Time-lapse observations (Fig. 1A and Movie S1) revealed that the morphology of almost all cells on the G5 surface was round with temporal stretching. After 3 days, these cells formed three-dimensional cell aggregates through coalescence between cells and aggregates. A remarkable change in shape with repeated extension and contraction of aggregates was occasionally observed, although the aggregates on G5 surface were loosely attached; these were relatively easy to collect after tapping of the culture vessels. However, cells on the PS surface were flat and demonstrated continuous stretching (Fig. 1B and Movie S2). Cells in round-bottom 96-well plates formed aggregates that did not adhere to the bottom of wells. These aggregates maintained a uniform size and shape over time (Fig. 1C). To evaluate the degree of cardiomyocyte differentiation, single cells suspension of hMSCs were re-seeded on the PS surface after harvesting, and identified by immunofluorescent staining for cTnT. Fluorescent microscopy (Fig. 2) revealed two main types of cells cultured on G5 surface: small spindle-like and large polygonal cells. Staining for cTnT and F-actin revealed thin filaments corresponding to cTnT in the cytoplasm of the large polygonal cells. This pattern was not observed in cells with spindle-like cells. To examine cell aggregates formed in response to environmental cues, time profiles of cell growth and differentiation were
2.3. Determination of cell growth and differentiation of aggregates To determine the cell number (XT ), cells were enzymatically detached using a 0.1% trypsin/0.02% EDTA solution, followed by direct counting of suspended cells using trypan blue exclusion on a hemocytometer. To analyze hMSC differentiation into cardiomyocytes, cells cultured for various periods were stained with a nuclear stain and
Fig. 1. Morphological characterization of hMSCs cultured on the G5 surface (A), PS surface (B), and round-bottom 96-well plate (C). Scale bars indicate 100 m.
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Fig. 2. Fluorescent images showing cardiac troponin T (cTnT; green), F-actin (red) and nuclei (blue) of cells cultured on the G5 surface (A), PS surface (B), and round-bottom 96-well plate (C) for t = 10 days. Scale bars indicate 50 m. The images in panels A1 (left) and A2 (right) are higher magnifications of the boxed areas in panel A. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
A
0.3
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0.1
0
B
6
4
2
0
0
obtained by multiplying the XT /X0 and the XP /XT , was 0.55 on the G5 surface, which was higher than that on PS surface. To confirm the presence of cTnT+ cells on the G5 surface, we quantitatively evaluated the XP /XT value of two populations: cells in collected aggregates; and the cells remaining on the G5 surface after tapping. As shown in Fig. 4, the XP /XT in collected aggregates from the G5 surface was 0.31 at t = 10 days, which was 1.4-fold higher than for XP /XT of cells remaining on the culture vessel after tapping. These results concluded that the highest level of in cells in collected aggregates, compared to that remaining cells, suggesting that the aggregation process on the G5 surface in a dynamic manner leads to lineage commitment toward a cardiomyogenic fate in vitro. To examine the mechanism of cell aggregation and cardiomyogenic potential associated with active migration on G5 surface, the hMSC cultures were performed by using culture medium with and without exposure to 10 M Rac1 inhibitor (NSC23766) after t = 3 days. We found that the cells with exposure to Rac1 inhibitor reduced the aggregate formation at t = 10 days (Fig. S1), compared to those without exposure. The XP /XT in culture with exposure significantly decreased to be 0.15 at t = 10 days, compared to that without exposure although there are no significant difference between the XT /X0 in cultures with and without exposure (Fig. S2). These results suggest that the cells exposed to Rac1 inhibitor reduced the cardiomyogenic potential due to less reduced aggregate formation during culture.
Ratio of cTnT positive cells, XP/XT (-)
Ratio of total cell number, XT/X0 (-)
Ratio of cTnT positive cells, XP/XT (-)
compared under three conditions. As shown in Fig. 3, the ratio of adherent cells to seeded cells (XT /X0 ) on the G5 surface gradually increased over time, reaching 2.5 at t = 10 days. The XT /X0 corresponding to the G5 surface was less than half the level observed for the PS surface. For cell aggregates maintained on round-bottom 96-well plates, the XT /X0 was approximately 1. The ratio of cTnT+ cells to adherent cells (XP /XT ) on the G5 surface at t = 3 days was low, similar to that on the PS surface. At t = 5 days, XP /XT was markedly increased in culture on the G5 surface, and the level of XP /XT on the G5 surface plateaued to 0.22 at t = 10 days, which was 3.2-fold higher than on PS surface. The XP /XT on the round-bottom 96-well plate was still at a low level during culture. At t = 10 days, the total cell number of cTnT+ cells (XP /X0 ), which was
5
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Culture time, t (days) Fig. 3. Proliferation and cardiomyogenic potential of hMSCs cultured under various conditions. Time profiles of XT /X0 (A) and XP /XT (B) evaluated over 10 days. Symbols: closed triangle, PS surface; closed circles, G5 surface; and open circles, round-bottom 96-well plate. Vertical bars indicate standard deviations (n = 3).
(*p< 0.01)
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0
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Fig. 4. Cardiomyogenic potential of hMSC aggregates cultured on the G5 surface for 10 days. Cultures were conducted on the G5 surface and then divided into two populations. Cells in collected aggregates (A) and cells remaining on the culture vessel (B) by tapping at t = 10 days without trypsinization. XP /XT evaluated for the two populations. Vertical bars indicate the standard deviations (n = 3).
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Fig. 5. Dynamic cell behavior and cardiomyogenic potential of hMSCs after reseeding of cell aggregates on the G5 surface after 10 days. (A) Cell behavior in representative cell aggregates after re-seeding on the G5 and PS surfaces. Cell aggregates that loosely attached to the G5 surface were harvested by tapping after culturing for 10 days, and then re-seeded on the G5 and PS surfaces. Scale bars indicate 100 m. The images in boxed areas for the panels show dynamic morphological changes and division of the traced cells at t = 14.7 days. (B) XT /X0 and XP /XT values were evaluated for cells cultured on G5 surface over 20 days after passaging cell aggregates for 10 days. Vertical bars indicate the standard deviations (n = 3).
3.2. Enrichment of cardiomyocyte-like cells through aggregate passage on G5 surface Cell aggregates cultured on the G5 surface for 10 days were collected by tapping, and the harvested aggregates were re-seeded onto the G5 and PS surfaces. Fig. 5A shows representative images of aggregates after re-seeding onto the G5 and PS surfaces. When the cell aggregates were subcultured on G5 surface, the aggregates adhered to the surface and initiated to disperse as single cells migrated out actively from cell aggregates, and these cells repeatedly stretched and contracted (Movie S3). Prolonged incubation resulted in active cell migration with reformation and coalescence among aggregates. On the other hand, cell aggregates subcultured on PS surface adhered and spread without any dispersion. After further incubation, the aggregates kept flat shape without any reformation into spherical one (Movie S4). As shown in Fig. 5B, XT /X0 values were not significantly different between aggregates after re-seeding onto the G5 and PS surfaces. However, the XP /XT value for aggregate on G5 surface at t = 20 days showed higher level of XP /XT = 0.39, compared to that of aggregates on PS surface, suggesting this aggregate passage causes the increment in population of cardiomyocyte-like cells. For long-term culture, two different passage methods were applied (Fig. 6A). For Run 1, cell aggregates attached to the G5 surface were harvested by tapping, and then re-seeded onto a freshly prepared G5 surface. For Run 2, single cells harvested from the G5 surface by enzymatic digestion were seeded onto freshly prepared G5 surface.
Fig. 6. Enrichment of cardiomyocyte-like cells derived from hMSC by passaging cell aggregates on the G5 surface. (A) Schematic showing the hMSC culture process. For Run 1, cells were cultured on the G5 surface for 40 days. Cells were subcultured every 10 days by tapping to collect cell aggregates. For Run 2, conventional culture protocols were used and cells were trypsinized to facilitate dissociation and collection. (B) Time profiles of XP /XT values were determined for 40 days. Symbols: close circle, at end of first passage (10 days); open circles, Run 1; and open triangle, Run 2.
Long-term culturing was conducted for 40 days after the third passage, to increase the frequency of cTnT+ cells. During long-term culture (Fig. 6B), the XP /XT in cultures gradually increased for both methods. For Run 1, the XP /XT value on the G5 surface increased to be 0.62 at the third passage, 1.3-fold higher than that for Run 2. These results suggested that the aggregate formation acted on the fate determination toward a cardiomyogenic lineage and the passage of cells on G5 surface promoted the induction. 4. Discussion For a differentiation strategy to be useful in clinical applications, directing fate determination and differentiation of stem cells is critical. When a stem cell is committed or specified, the final differentiated phenotype of the cell is not yet determined, and any bias the cell has toward a certain fate can be altered or even reversed. In this study, we highlight how stem cells proliferate or commit toward a cardiomyocyte lineage, their distinct properties, and how this information can be used to develop novel strategies for regulating fate determination. Waddington described cellular differentiation as a ball rolling through an epigenetic landscape [12–16]. Once determined, cell fate is further specified only through maturation process, which includes differentiation and senescence. Waddington’s report suggests that a cell, in response to an energy landscape, determines its differentiation direction from activation of a particular “instructive” pathway, or a consecutive series of specific genes. However, recent studies have shown that it is possible
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to influence cell fate through artificial manipulation, and the exogenous expression of molecular mechanisms underlying epigenetics has led to the realization that, through adding or withdrawing certain cytokines and/or growth factors and through changing the transcriptional activity of genes, cells can be dedifferentiated and refocused to different lineages. Using such a paradigm is of significance to stem cell bioengineering because it breaks down the ultimate goal of precisely directing the fates of stem cells during the early-state differentiation. 4.1. Aggregate formation of hMSCs in a dynamic manner induces initiation of lineage commitment toward a cardiomyogenic fate In this study, we have described alternative approaches that significantly influence fate determination toward a cardiomyogenic lineage, thereby resulting in a greater number of cardiomyocytelike cells. Directing of stem cell fate is thought to be vital in producing a homogeneous population of cardiomyocytes. The formation of aggregates is an important event influencing the fate of cultured stem cells, and the mechanisms of aggregate formation in vitro need to be elucidated to understand cellular differentiation. In comparison with conventional monolayer cultures, multicellular aggregate closely resembles the situation in vivo with regard to differentiation patterns, spatial cell-cell interactions and extracellular matrix interactions [17–21]. Several reports have described the major roles of cadherins during aggregate formation [18]. In the present study, we examined the influence of cell aggregation on hMSC differentiation. Time-lapse observations showed that cells grown on the G5 surface exhibited active migration, and were associated with periodic changes in cell morphology. As the culture period was extended, the majority of cells on the G5 surface aggregated through cell division and coalescence of migrating cells (Movie S1). In addition, the inhibitory experiment using Rac1 inhibitor (Fig. S2) reveals the importance of migration for aggregate formation. The cell aggregates on G5 surface showed more dynamic changes in morphology associated with active “self-stretching” of aggregates during migration. In contrast, hMSCs spontaneously formed floating multicellular aggregates in conventional cultures within a short adaptation period (24 h), owing to the absence of binding sites for integrin mediation (Fig. 1C). The difference in cell aggregate formation resulted in varied properties between active attached aggregate (motile and multiplying) and passive unattached aggregates (non-motile and non-growing). For analysis of cardiomyogenic differentiation, the thin filament structure of cTnT was detected throughout the cytoplasm of cells within aggregates grown on the G5 surface at 10 days (Fig. 2A1). The proportion of cTnT+ cells in the aggregates was 3.2-folder higher than that in monolayer cells on the PS surface. It is worth noting that passive cell aggregates consisting of spindle-shaped cells demonstrated the expression of cTnT. From these finding, it is most likely that the formation of cell aggregates in a dynamic manner on the G5 surface encourage fate determination toward a cardiomyocyte-like cell. 4.2. Repeated collapse and formation of aggregates enhanced hMSC populations guiding them toward a cardiomyogenic fate Cell fate determination in the stem cells is regulated by many microenvironmental cues and intracellular signals, with changes in cell aggregate morphology likely the most important among theses. Cell aggregation occurred through a balance between cell–cell and cell–substrate adhesions. Regulation of the balance between cell–cell and cell–substrate adhesions by active cell migration leads to different fates. In this study, culturing cell aggregate on G5 surface revealed that repeated disruption and formation of aggregates enhanced guidance of the hMSC population toward a cardiomyocyte fate (Figs. 5 and 6). However, the population of
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cardiomyocyte-like cells within cultured aggregates on the G5 surface over 10 days was not increased (Fig. 3). These had been thought to form floating aggregates, which are detached from the substratum through the secretion of extracellular matrix components such as collagen, proteoglycans and fibronectin, by resident cells. In cell aggregates subcultured on the new G5 surface, undifferentiated cells migrated out from the aggregate and began to undergo morphological changes. The migratory behaviors of cells on the G5 surface promoted reformation of aggregates through cell division and coalescence between cells or aggregates. The major event for commitment toward a cardiomyogenic fate is thought to be the process of aggregate formation, accompanied by cell migration with extension and contraction on dendrimer surface. The cells in the aggregate spontaneously disperse following re-seeding of the aggregates seeded on a new dendrimer surface, leading to increased numbers of cTnT+ cells. Cell aggregates passaged three times over 40 days, on the G5 surface expressed cTnT. These results suggest that expanding aggregates from the third passage on the G5 surface results in an enriched population of cardiomyocyte-like cells. In our previous study, we described the changes in cell morphology as well as the potentials for anchorage and migration on the G5 surface [9]. Immunostainings of F-actin, Rac1, and N-cadherin showed the Rac1 up-regulation on the leading edges of cells with lamellipodia when cultured on the G5 surface. As cell–cell contact formation proceeded, the cells results in the formation of cell aggregates. It is most likely that the N-cadherin expression in the mound-shaped cells was regulated in a spatiotemporal manner, supporting that an increase in N-cadherin-dependent cell-cell contacts promotes the formation of cell aggregates on the G5 surface. This suggests that high Rac1 activity leads to the lamellipodium formation that contributes to the aggregate formation through Ncadherin-mediated cell–cell contacts. In addition, cell aggregation associated with dynamic cell migration caused an increase in Wnt signaling, thereby regulating hMSC differentiation into cardiomyogenic cells [9]. Differentiation of hMSCs toward cell lineages such as chondrocytes, adipocytes and osteoblasts was not detected within cell aggregates grown on G5 surface although partial expression of the pluripotency marker CD 105 was observed. Although a detailed mechanism underlying cell migration properties within cell aggregates on the G5 surface remains unclear, a consequence of this migration is up-regulated expression of the integrin and Wnt/catenin signaling pathway, which are crucial for cardiomyogenesis [22,23]. Lee and Heo demonstrated the stimulating effects of cyclic strain on cardiomyogenesis from embryonic stem cells. This occurs through cooperative signaling between Wnt and integrin-linked kinase within the signaling cascade for regulating cardiomyogenesis [23]. The findings from our study suggest that cyclic mechanical strains, generated in the cytoskeleton of cells, and exerted upon culture surfaces and neighboring cells, play a central role in the induction of a cardiomyocyte-like cell population within aggregates.
5. Conclusions Our results suggest a novel culture strategy for inducing early lineage commitment of hMSCs toward a cardiomyogenic fate. Encouraging migration of cells on the G5 surface appears to assist the formation of aggregates, resulting in the enhanced efficiency of early lineage commitment of hMSCs toward a cardiomyogenic fate. Further passage of all aggregates without enzymatic digestion increased the population of cardiomyocyte-like cells. A strategy incorporating interactions between cells and soluble factors can offer an alternative approach for the control of lineage direction and specification. This methodology would provide easy access to a large number of homogeneous differentiated cells, and allow
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for the development of more effective procedures with respect to expansion and differentiation of stem cells into cardiomyocytes. Acknowledgements This work was partially supported by the Japan Science and Technology Agency (JST), Grants-in-Aid for Scientific Research (B) (no. 21360402) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by the MEXT project, “Creating Hybrid Organs of the future” at Osaka University, and by the Japan Society for the Promotion of Science (JSPS) Japanese-German Graduate Externship. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2013.12.015. References [1] P. Bianco, M. Riminucci, S. Gronthos, P.G. Robey, Bone marrow stromal stem cells: nature, biology, and potential applications, Stem Cells 19 (2001) 180–192. [2] R. Peerani, P.W. Zandstra, Enabling stem cell therapies through synthetic stem cell-niche engineering, J. Clin. Invest. 120 (2010) 60–70. [3] D. Schaffer, Exploring and engineering stem cells and their niches, Curr. Opin. Chem. Biol. 11 (2007) 355–356. [4] G.C. Reilly, A.J. Engler, Intrinsic extracellular matrix properties regulate stem cell differentiation, J. Biomech. 43 (2010) 55–62. [5] Kshitiz, D.-H. Kim, D.J. Beebe, A. Levchenko, Micro- and nanoengineering for stem cell biology: the promise with a caution, Trends Biotechnol. 29 (2011) 399–408. [6] E. Ghafar-Zadeh, J.R. Waldeisen, L.P. Lee, Engineered approaches to the stem cell microenvironment for cardiac tissue regeneration, Lab Chip 11 (2011) 3031–3048. [7] Y. Zhang, Y. Chu, W. Shen, Z. Dou, Effect of 5-azacytidine induction duration on differentiation of human first-trimester fetal mesenchymal stem cells towards cardiomyocyte-like cells, Interact. Cardiovasc. Thorac. Surg. 9 (2009) 943–946. [8] M.-H. Kim, M. Kino-oka, M. Taya, Designing culture surfaces based on cell anchoring mechanisms to regulate cell morphologies and functions, Biotechnol. Adv. 28 (2010) 7–16. [9] M.-H. Kim, M. Kino-oka, N. Maruyama, A. Saito, Y. Sawa, M. Taya, Cardiomyogenic induction of human mesenchymal stem cells by altered Rho family GTPase expression on dendrimer-immobilized surface with d-glucose display, Biomaterials 31 (2010) 7666–7677.
[10] S. Mashayekhan, M.-H. Kim, S. Miyazaki, F. Tashiro, M. Kino-oka, M. Taya, J. Miyazaki, Enrichment of undifferentiated mouse embryonic stem cells on a culture surface with a glucose-displaying dendrimer, Biomaterials 29 (2008) 4236–4243. [11] M.-H. Kim, M. Kino-oka, Y. Morinaga, Y. Sawada, M. Kawase, K. Yagi, M. Taya, Morphological regulation and aggregate formation of rabbit chondrocytes on dendrimer-immobilized surfaces with d-glucose display, J. Biosci. Bioeng. 107 (2009) 196–205. [12] J. Wang, L. Xu, E.K. Wang, S. Huang, The potential landscape of genetic circuits imposes the arrow of time in stem cell differentiation, Biophys. J. 99 (2010) 29–39. [13] J. Wang, K. Zhang, L. Xu, E. Wang, Quantifying the Waddington landscape and biological paths for development and differentiation, Proc. Natl. Acad. Sci. U.S.A. 108 (2011) 8257–8262. [14] J.X. Zhou, S. Huang, Understanding gene circuits at cell-fate branch points for rational cell reprogramming, Trends Genet. 27 (2011) 55–62. [15] S. Bhattacharya, Q. Zhang, M.E. Andersen, A deterministic map of Waddington’s epigenetic landscape for cell fate specification, BMC Syst. Biol. 5 (2011) 85. [16] M. Hemberger, W. Dean, W. Reik, Epigenetic dynamics of stem cells and cell lineage commitment: digging Waddington’s canal, Nat. Rev. Mol. Cell Biol. 10 (2009) 526–537. [17] I.A. Potapova, P.R. Brink, I.S. Cohen, S.V. Doronin, Culturing of human mesenchymal stem cells as three-dimensional aggregates induces functional expression of CXCR4 that regulates adhesion to endothelial cells, J. Biol. Chem. 283 (2008) 13100–13107. [18] C.C. Wang, C.H. Chen, S.M. Hwang, W.W. Lin, C.H. Huang, W.Y. Lee, Y. Chang, H.W. Sung, Spherically symmetric mesenchymal stromal cell bodies inherent with endogenous extracellular matrices for cellular cardiomyoplasty, Stem Cells 27 (2009) 724–732. [19] G.-S. Huang, L.-G. Dai, L.B. Yen, S.-H. Hsu, Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes, Biomaterials 32 (2011) 6929–6945. [20] W. Wang, K. Itaka, S. Ohba, N. Nishiyama, U.I. Chung, Y. Yamasaki, K. Kataoka, 3D spheroid culture system on micropatterned substrates for improved differentiation efficiency of multipotent mesenchymal stem cells, Biomaterials 30 (2009) 2705–2715. [21] T.J. Bartosh, J.H. Ylöstalo, A. Mohammadipoor, N. Bazhanov, K. Coble, K. Claypool, R.H. Lee, H. Choi, D.J. Prockop, Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their anti-inflammatory properties, Proc. Natl. Acad. Sci. U.S.A. 107 (2010) 13724–13729. [22] A. Salameh, A. Wustmann, S. Karl, K. Blanke, D. Apel, D. Rojas-Gomez, H. Franke, F.W. Mohr, J. Janousek, S. Dhein, Cyclic mechanical stretch induces cardiomyocyte orientation and polarization of the gap junction protein connexin43, Circ. Res. 106 (2010) 1592–1602. [23] J.S. Heo, J.C. Lee, -Catenin mediates cyclic strain-stimulated cardiomyogenesis in mouse embryonic stem cells through ROS-dependent and integrin-mediated PI3K/Akt pathways, J. Cell Biochem. 112 (2011) 1880–1889.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular Article
Directed differentiation of human mesenchymal stem cells toward a cardiomyogenic fate commitment through formation of cell aggregates夽 Mee-Hae Kim a , Yuuki Ogawa a , Koji Yamada b , Masahito Taya b , Masahiro Kino-oka a,∗ a b
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan
a r t i c l e
i n f o
Article history: Received 16 August 2013 Received in revised form 9 November 2013 Accepted 23 December 2013 Available online 15 January 2014 Keywords: Animal cell culture Growth kinetics Tissue cell culture Biomedical Human mesenchymal stem cells Cardiomyogenic fate Dendrimer surface
a b s t r a c t Cell morphology is known to modulate the multipotential lineage commitment of stem cells. We provide a new strategy to induce the early lineage commitment of human mesenchymal stem cells (hMSCs) toward a cardiomyogenic fate through the formation of cell aggregates. A surface-immobilized polyamidoamine dendrimer with fifth generation of dendron structure was used during the culturing of hMSCs. These hMSCs cultured on the G5 surface formed aggregates through active migration and division. More than 22% of cardiac troponin-T (cTnT)-positive (cTnT+ ) cells in aggregates formed on the dendrimer surface; the population formed on the dendrimer surface was higher than that in conventional culture vessel. When cell aggregate was reseeded onto a fresh G5 surface, single cells migrated out of the aggregates, proliferated, and formed new aggregates. This passage method, accompanied with repetitive aggregate dispersion and formation, was applied to cultures over 40 days. The proportion of cTnT+ cells increased to 62% by the end of third passage. Our results suggest that culturing hMSCs on G5 surface results in directed commitment of the hMSCs toward a cardiomyocyte-like fate. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Mesenchymal stem cells (MSCs) are attracting increased attention because they possess multi-lineage developmental potential and the capacity for self-renewal in vivo [1,2]. MSCs are multipotent cells, initially isolated from the bone marrow, and are noted for their ability to differentiate into osteoblasts, chondrocytes, and adipocytes [2]. As an autologous source of stem cells, MSCs have been considered a possible source of cells that can be applied to regenerative therapies [1]. To facilitate in vitro development, many researchers have attempted to design microenvironments that mimic the stem cell niche, thereby driving cells down their preferred differentiation pathway [3–5]. The niche paradigm outlines three interactions involving cells, culture medium components, adjacent cells and substrates; these are cell-soluble factor, cell–cell, and cell–substrate interactions [3]. In vitro strategies for regulating stem cell behavior attempt to mimic developmental mechanisms and establish artificial environments that promote specific cell fates [2,5]. The majority of previous research studies have focused on dynamic process that are tightly orchestrated by the sequential
夽 Much of this work forms the basis of the Ph.D. dissertation of Yuuki Ogawa. ∗ Corresponding author. Tel.: +81 06 6879 7444; fax: +81 06 6879 4246. E-mail address: [email protected] (M. Kino-oka). 1369-703X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.12.015
expression of multiple signal transduction proteins and transcription factors working in a combination [4,6]. A number of signaling pathways (Wnts/Nodal) and growth factors (bone morphogenic proteins and fibroblast growth factor) have been implicated in the development of specialized cardiac subtypes [4–6]. The times and concentrations at which these factors affect differentiation toward the desired fate have been optimized. Although human MSCs (hMSCs) possess a self-renewing capacity, they display heterogeneous responses upon the induction of differentiation, resulting in a mixture of differentiated cells [5,6]. Zhang et al. [7] reported that culturing MSCs for 21 days with 5-azacytidine in culture medium induced 45% cardiomyocyte-like cells that were cardiac troponin-T positive (cTnT+ ); however, other differentiation pathways were also followed leading to a heterogeneous population of differentiated cells. Fully functional cardiomyocytes with striated cytoskeleton and proper electrical coupling for hMSCs have not been observed. Overall, it appears that the best differentiation strategies produce homogenous populations of cardiomyocytes; these strategies tend to be reproducible, result in cells of the appropriate quality, and produce large quantities of cells. In previous studies, the use of a dendrimer surface has been proposed as a method of regulating the morphology and function of cells [8–11]. Polyamidoamine dendrimers have been immobilized to culture surfaces, with the variations in generation number of the dendrimer surface shown to cause morphological changes in
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hMSCs. The observed morphological changes were accompanied by dynamic cytoskeletal formation in cells on the culture surface. Subsequent formation of cell aggregates led to cardiomyogenic differentiation of hMSCs in vitro [10]. It is believed that endogenous Rho family GTPase signaling pathways, accompanied by morphological changes, are an intrinsic cue for modulating transcription factors. In the present study, we used a fifth generation of dendrimer surface in an attempt to induce initiation of lineage commitment of hMSCs toward a cardiomyogenic fate. Novel approaches to cell culture including aggregate dispersion and formation during passage were trialed, and prolonged culturing was conducted to enhance the homogeneity of the cardiomyocyte-like cell population.
an antibody against cTnT (Materials and Methods in the Supporting Information). For quantitative assessment of cardiomyogenic differentiation, suspended cells were re-seeded after harvesting from the cultures as described above. The numbers of cTnT+ and the DAPI+ nuclei were evaluated to determine the ratio of cTnT+ (XP ) to DAPI+ (XT ) cells.
2. Materials and methods
3. Results
2.1. Cells and culture conditions
3.1. Dynamic behavior and spontaneous cardiomyogenic differentiation of hMSCs
Human bone marrow-derived MSCs were obtained from Lonza (Lot no. 8F3543; Walkersville, MD, USA). Routine subcultures of hMSCs were conducted in a 75-cm2 flask (Corning Costar, Cambridge, MA, USA) using hMSC growth medium (Lonza) at 37 ◦ C in a humidified atmosphere containing 5% CO2 . Upon reaching 70% confluence, cells were detached by enzymatic treatment with a 0.1% trypsin/0.02% EDTA solution (Sigma–Aldrich, St. Louis, MO, USA). Cells that had undergone less than five passages were used in subsequent experiments. For all experiments, hMSCs were expanded for the specified number of days in Dulbecco’s modified Eagle’s medium (DMEM; Sigma–Aldrich) supplemented with 10% fetal bovine serum (Invitrogen, Grand Island, NY, USA) and antibiotics (100 U/cm3 penicillin G, 0.1 mg/cm3 streptomycin and 0.25 mg/cm3 amphotericin B; Invitrogen). Cultures were grown in square 8-well plate with and without dendrimer immobilized to the surface. The seeding density was fixed at a viable cell concentration of 5.0 × 103 cells/cm2 (X0 ). Culture medium was replenished every 3 days. If necessary, round-bottomed 96-well plates were used to generate floating cell aggregates, which were used to prepare aggregates of hMSCs as controls. 2.2. Surface preparation The dendrimer surface was prepared using the conventional tissue culture polystyrene (PS) surface of a square 8-well plate (surface area; 10.5 cm2 , Nunc, Roskilde, Denmark), as described previous report [8]. Briefly, a G5 surface was created, under sterile conditions, by changing the generation number of synthesized dendrimers over four reactions. Hydroxyl groups were displayed on the plain surface by pouring potassium tert-butoxide into the wells. Then, aqueous glutaraldehyde was introduced into the wells, with the wells were then treated with a tris(2-aminoethyl) amine solution to produce a dendron structure. The wells were rinsed with sterile water and the previous step repeated until the fifth generation of dendrimers was synthesized. To display glucose as a terminal ligand, d-glucose was applied.
2.4. Statistical analysis All experiments were performed at least three times and data expressed as means with standard deviations. Student’s t-test was used to determine statistical significances among the data sets; pvalues less than 0.01 and 0.05 were considered significant.
Cultures of hMSCs were grown on G5 and PS surfaces in 8-well plates, and in round-bottom 96-well plates, with non-adhesive culture surfaces, for 10 days. Time-lapse observations (Fig. 1A and Movie S1) revealed that the morphology of almost all cells on the G5 surface was round with temporal stretching. After 3 days, these cells formed three-dimensional cell aggregates through coalescence between cells and aggregates. A remarkable change in shape with repeated extension and contraction of aggregates was occasionally observed, although the aggregates on G5 surface were loosely attached; these were relatively easy to collect after tapping of the culture vessels. However, cells on the PS surface were flat and demonstrated continuous stretching (Fig. 1B and Movie S2). Cells in round-bottom 96-well plates formed aggregates that did not adhere to the bottom of wells. These aggregates maintained a uniform size and shape over time (Fig. 1C). To evaluate the degree of cardiomyocyte differentiation, single cells suspension of hMSCs were re-seeded on the PS surface after harvesting, and identified by immunofluorescent staining for cTnT. Fluorescent microscopy (Fig. 2) revealed two main types of cells cultured on G5 surface: small spindle-like and large polygonal cells. Staining for cTnT and F-actin revealed thin filaments corresponding to cTnT in the cytoplasm of the large polygonal cells. This pattern was not observed in cells with spindle-like cells. To examine cell aggregates formed in response to environmental cues, time profiles of cell growth and differentiation were
2.3. Determination of cell growth and differentiation of aggregates To determine the cell number (XT ), cells were enzymatically detached using a 0.1% trypsin/0.02% EDTA solution, followed by direct counting of suspended cells using trypan blue exclusion on a hemocytometer. To analyze hMSC differentiation into cardiomyocytes, cells cultured for various periods were stained with a nuclear stain and
Fig. 1. Morphological characterization of hMSCs cultured on the G5 surface (A), PS surface (B), and round-bottom 96-well plate (C). Scale bars indicate 100 m.
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Fig. 2. Fluorescent images showing cardiac troponin T (cTnT; green), F-actin (red) and nuclei (blue) of cells cultured on the G5 surface (A), PS surface (B), and round-bottom 96-well plate (C) for t = 10 days. Scale bars indicate 50 m. The images in panels A1 (left) and A2 (right) are higher magnifications of the boxed areas in panel A. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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obtained by multiplying the XT /X0 and the XP /XT , was 0.55 on the G5 surface, which was higher than that on PS surface. To confirm the presence of cTnT+ cells on the G5 surface, we quantitatively evaluated the XP /XT value of two populations: cells in collected aggregates; and the cells remaining on the G5 surface after tapping. As shown in Fig. 4, the XP /XT in collected aggregates from the G5 surface was 0.31 at t = 10 days, which was 1.4-fold higher than for XP /XT of cells remaining on the culture vessel after tapping. These results concluded that the highest level of in cells in collected aggregates, compared to that remaining cells, suggesting that the aggregation process on the G5 surface in a dynamic manner leads to lineage commitment toward a cardiomyogenic fate in vitro. To examine the mechanism of cell aggregation and cardiomyogenic potential associated with active migration on G5 surface, the hMSC cultures were performed by using culture medium with and without exposure to 10 M Rac1 inhibitor (NSC23766) after t = 3 days. We found that the cells with exposure to Rac1 inhibitor reduced the aggregate formation at t = 10 days (Fig. S1), compared to those without exposure. The XP /XT in culture with exposure significantly decreased to be 0.15 at t = 10 days, compared to that without exposure although there are no significant difference between the XT /X0 in cultures with and without exposure (Fig. S2). These results suggest that the cells exposed to Rac1 inhibitor reduced the cardiomyogenic potential due to less reduced aggregate formation during culture.
Ratio of cTnT positive cells, XP/XT (-)
Ratio of total cell number, XT/X0 (-)
Ratio of cTnT positive cells, XP/XT (-)
compared under three conditions. As shown in Fig. 3, the ratio of adherent cells to seeded cells (XT /X0 ) on the G5 surface gradually increased over time, reaching 2.5 at t = 10 days. The XT /X0 corresponding to the G5 surface was less than half the level observed for the PS surface. For cell aggregates maintained on round-bottom 96-well plates, the XT /X0 was approximately 1. The ratio of cTnT+ cells to adherent cells (XP /XT ) on the G5 surface at t = 3 days was low, similar to that on the PS surface. At t = 5 days, XP /XT was markedly increased in culture on the G5 surface, and the level of XP /XT on the G5 surface plateaued to 0.22 at t = 10 days, which was 3.2-fold higher than on PS surface. The XP /XT on the round-bottom 96-well plate was still at a low level during culture. At t = 10 days, the total cell number of cTnT+ cells (XP /X0 ), which was
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Culture time, t (days) Fig. 3. Proliferation and cardiomyogenic potential of hMSCs cultured under various conditions. Time profiles of XT /X0 (A) and XP /XT (B) evaluated over 10 days. Symbols: closed triangle, PS surface; closed circles, G5 surface; and open circles, round-bottom 96-well plate. Vertical bars indicate standard deviations (n = 3).
(*p< 0.01)
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Fig. 4. Cardiomyogenic potential of hMSC aggregates cultured on the G5 surface for 10 days. Cultures were conducted on the G5 surface and then divided into two populations. Cells in collected aggregates (A) and cells remaining on the culture vessel (B) by tapping at t = 10 days without trypsinization. XP /XT evaluated for the two populations. Vertical bars indicate the standard deviations (n = 3).
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Fig. 5. Dynamic cell behavior and cardiomyogenic potential of hMSCs after reseeding of cell aggregates on the G5 surface after 10 days. (A) Cell behavior in representative cell aggregates after re-seeding on the G5 and PS surfaces. Cell aggregates that loosely attached to the G5 surface were harvested by tapping after culturing for 10 days, and then re-seeded on the G5 and PS surfaces. Scale bars indicate 100 m. The images in boxed areas for the panels show dynamic morphological changes and division of the traced cells at t = 14.7 days. (B) XT /X0 and XP /XT values were evaluated for cells cultured on G5 surface over 20 days after passaging cell aggregates for 10 days. Vertical bars indicate the standard deviations (n = 3).
3.2. Enrichment of cardiomyocyte-like cells through aggregate passage on G5 surface Cell aggregates cultured on the G5 surface for 10 days were collected by tapping, and the harvested aggregates were re-seeded onto the G5 and PS surfaces. Fig. 5A shows representative images of aggregates after re-seeding onto the G5 and PS surfaces. When the cell aggregates were subcultured on G5 surface, the aggregates adhered to the surface and initiated to disperse as single cells migrated out actively from cell aggregates, and these cells repeatedly stretched and contracted (Movie S3). Prolonged incubation resulted in active cell migration with reformation and coalescence among aggregates. On the other hand, cell aggregates subcultured on PS surface adhered and spread without any dispersion. After further incubation, the aggregates kept flat shape without any reformation into spherical one (Movie S4). As shown in Fig. 5B, XT /X0 values were not significantly different between aggregates after re-seeding onto the G5 and PS surfaces. However, the XP /XT value for aggregate on G5 surface at t = 20 days showed higher level of XP /XT = 0.39, compared to that of aggregates on PS surface, suggesting this aggregate passage causes the increment in population of cardiomyocyte-like cells. For long-term culture, two different passage methods were applied (Fig. 6A). For Run 1, cell aggregates attached to the G5 surface were harvested by tapping, and then re-seeded onto a freshly prepared G5 surface. For Run 2, single cells harvested from the G5 surface by enzymatic digestion were seeded onto freshly prepared G5 surface.
Fig. 6. Enrichment of cardiomyocyte-like cells derived from hMSC by passaging cell aggregates on the G5 surface. (A) Schematic showing the hMSC culture process. For Run 1, cells were cultured on the G5 surface for 40 days. Cells were subcultured every 10 days by tapping to collect cell aggregates. For Run 2, conventional culture protocols were used and cells were trypsinized to facilitate dissociation and collection. (B) Time profiles of XP /XT values were determined for 40 days. Symbols: close circle, at end of first passage (10 days); open circles, Run 1; and open triangle, Run 2.
Long-term culturing was conducted for 40 days after the third passage, to increase the frequency of cTnT+ cells. During long-term culture (Fig. 6B), the XP /XT in cultures gradually increased for both methods. For Run 1, the XP /XT value on the G5 surface increased to be 0.62 at the third passage, 1.3-fold higher than that for Run 2. These results suggested that the aggregate formation acted on the fate determination toward a cardiomyogenic lineage and the passage of cells on G5 surface promoted the induction. 4. Discussion For a differentiation strategy to be useful in clinical applications, directing fate determination and differentiation of stem cells is critical. When a stem cell is committed or specified, the final differentiated phenotype of the cell is not yet determined, and any bias the cell has toward a certain fate can be altered or even reversed. In this study, we highlight how stem cells proliferate or commit toward a cardiomyocyte lineage, their distinct properties, and how this information can be used to develop novel strategies for regulating fate determination. Waddington described cellular differentiation as a ball rolling through an epigenetic landscape [12–16]. Once determined, cell fate is further specified only through maturation process, which includes differentiation and senescence. Waddington’s report suggests that a cell, in response to an energy landscape, determines its differentiation direction from activation of a particular “instructive” pathway, or a consecutive series of specific genes. However, recent studies have shown that it is possible
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to influence cell fate through artificial manipulation, and the exogenous expression of molecular mechanisms underlying epigenetics has led to the realization that, through adding or withdrawing certain cytokines and/or growth factors and through changing the transcriptional activity of genes, cells can be dedifferentiated and refocused to different lineages. Using such a paradigm is of significance to stem cell bioengineering because it breaks down the ultimate goal of precisely directing the fates of stem cells during the early-state differentiation. 4.1. Aggregate formation of hMSCs in a dynamic manner induces initiation of lineage commitment toward a cardiomyogenic fate In this study, we have described alternative approaches that significantly influence fate determination toward a cardiomyogenic lineage, thereby resulting in a greater number of cardiomyocytelike cells. Directing of stem cell fate is thought to be vital in producing a homogeneous population of cardiomyocytes. The formation of aggregates is an important event influencing the fate of cultured stem cells, and the mechanisms of aggregate formation in vitro need to be elucidated to understand cellular differentiation. In comparison with conventional monolayer cultures, multicellular aggregate closely resembles the situation in vivo with regard to differentiation patterns, spatial cell-cell interactions and extracellular matrix interactions [17–21]. Several reports have described the major roles of cadherins during aggregate formation [18]. In the present study, we examined the influence of cell aggregation on hMSC differentiation. Time-lapse observations showed that cells grown on the G5 surface exhibited active migration, and were associated with periodic changes in cell morphology. As the culture period was extended, the majority of cells on the G5 surface aggregated through cell division and coalescence of migrating cells (Movie S1). In addition, the inhibitory experiment using Rac1 inhibitor (Fig. S2) reveals the importance of migration for aggregate formation. The cell aggregates on G5 surface showed more dynamic changes in morphology associated with active “self-stretching” of aggregates during migration. In contrast, hMSCs spontaneously formed floating multicellular aggregates in conventional cultures within a short adaptation period (24 h), owing to the absence of binding sites for integrin mediation (Fig. 1C). The difference in cell aggregate formation resulted in varied properties between active attached aggregate (motile and multiplying) and passive unattached aggregates (non-motile and non-growing). For analysis of cardiomyogenic differentiation, the thin filament structure of cTnT was detected throughout the cytoplasm of cells within aggregates grown on the G5 surface at 10 days (Fig. 2A1). The proportion of cTnT+ cells in the aggregates was 3.2-folder higher than that in monolayer cells on the PS surface. It is worth noting that passive cell aggregates consisting of spindle-shaped cells demonstrated the expression of cTnT. From these finding, it is most likely that the formation of cell aggregates in a dynamic manner on the G5 surface encourage fate determination toward a cardiomyocyte-like cell. 4.2. Repeated collapse and formation of aggregates enhanced hMSC populations guiding them toward a cardiomyogenic fate Cell fate determination in the stem cells is regulated by many microenvironmental cues and intracellular signals, with changes in cell aggregate morphology likely the most important among theses. Cell aggregation occurred through a balance between cell–cell and cell–substrate adhesions. Regulation of the balance between cell–cell and cell–substrate adhesions by active cell migration leads to different fates. In this study, culturing cell aggregate on G5 surface revealed that repeated disruption and formation of aggregates enhanced guidance of the hMSC population toward a cardiomyocyte fate (Figs. 5 and 6). However, the population of
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cardiomyocyte-like cells within cultured aggregates on the G5 surface over 10 days was not increased (Fig. 3). These had been thought to form floating aggregates, which are detached from the substratum through the secretion of extracellular matrix components such as collagen, proteoglycans and fibronectin, by resident cells. In cell aggregates subcultured on the new G5 surface, undifferentiated cells migrated out from the aggregate and began to undergo morphological changes. The migratory behaviors of cells on the G5 surface promoted reformation of aggregates through cell division and coalescence between cells or aggregates. The major event for commitment toward a cardiomyogenic fate is thought to be the process of aggregate formation, accompanied by cell migration with extension and contraction on dendrimer surface. The cells in the aggregate spontaneously disperse following re-seeding of the aggregates seeded on a new dendrimer surface, leading to increased numbers of cTnT+ cells. Cell aggregates passaged three times over 40 days, on the G5 surface expressed cTnT. These results suggest that expanding aggregates from the third passage on the G5 surface results in an enriched population of cardiomyocyte-like cells. In our previous study, we described the changes in cell morphology as well as the potentials for anchorage and migration on the G5 surface [9]. Immunostainings of F-actin, Rac1, and N-cadherin showed the Rac1 up-regulation on the leading edges of cells with lamellipodia when cultured on the G5 surface. As cell–cell contact formation proceeded, the cells results in the formation of cell aggregates. It is most likely that the N-cadherin expression in the mound-shaped cells was regulated in a spatiotemporal manner, supporting that an increase in N-cadherin-dependent cell-cell contacts promotes the formation of cell aggregates on the G5 surface. This suggests that high Rac1 activity leads to the lamellipodium formation that contributes to the aggregate formation through Ncadherin-mediated cell–cell contacts. In addition, cell aggregation associated with dynamic cell migration caused an increase in Wnt signaling, thereby regulating hMSC differentiation into cardiomyogenic cells [9]. Differentiation of hMSCs toward cell lineages such as chondrocytes, adipocytes and osteoblasts was not detected within cell aggregates grown on G5 surface although partial expression of the pluripotency marker CD 105 was observed. Although a detailed mechanism underlying cell migration properties within cell aggregates on the G5 surface remains unclear, a consequence of this migration is up-regulated expression of the integrin and Wnt/catenin signaling pathway, which are crucial for cardiomyogenesis [22,23]. Lee and Heo demonstrated the stimulating effects of cyclic strain on cardiomyogenesis from embryonic stem cells. This occurs through cooperative signaling between Wnt and integrin-linked kinase within the signaling cascade for regulating cardiomyogenesis [23]. The findings from our study suggest that cyclic mechanical strains, generated in the cytoskeleton of cells, and exerted upon culture surfaces and neighboring cells, play a central role in the induction of a cardiomyocyte-like cell population within aggregates.
5. Conclusions Our results suggest a novel culture strategy for inducing early lineage commitment of hMSCs toward a cardiomyogenic fate. Encouraging migration of cells on the G5 surface appears to assist the formation of aggregates, resulting in the enhanced efficiency of early lineage commitment of hMSCs toward a cardiomyogenic fate. Further passage of all aggregates without enzymatic digestion increased the population of cardiomyocyte-like cells. A strategy incorporating interactions between cells and soluble factors can offer an alternative approach for the control of lineage direction and specification. This methodology would provide easy access to a large number of homogeneous differentiated cells, and allow
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for the development of more effective procedures with respect to expansion and differentiation of stem cells into cardiomyocytes. Acknowledgements This work was partially supported by the Japan Science and Technology Agency (JST), Grants-in-Aid for Scientific Research (B) (no. 21360402) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by the MEXT project, “Creating Hybrid Organs of the future” at Osaka University, and by the Japan Society for the Promotion of Science (JSPS) Japanese-German Graduate Externship. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2013.12.015. References [1] P. Bianco, M. Riminucci, S. Gronthos, P.G. Robey, Bone marrow stromal stem cells: nature, biology, and potential applications, Stem Cells 19 (2001) 180–192. [2] R. Peerani, P.W. Zandstra, Enabling stem cell therapies through synthetic stem cell-niche engineering, J. Clin. Invest. 120 (2010) 60–70. [3] D. Schaffer, Exploring and engineering stem cells and their niches, Curr. Opin. Chem. Biol. 11 (2007) 355–356. [4] G.C. Reilly, A.J. Engler, Intrinsic extracellular matrix properties regulate stem cell differentiation, J. Biomech. 43 (2010) 55–62. [5] Kshitiz, D.-H. Kim, D.J. Beebe, A. Levchenko, Micro- and nanoengineering for stem cell biology: the promise with a caution, Trends Biotechnol. 29 (2011) 399–408. [6] E. Ghafar-Zadeh, J.R. Waldeisen, L.P. Lee, Engineered approaches to the stem cell microenvironment for cardiac tissue regeneration, Lab Chip 11 (2011) 3031–3048. [7] Y. Zhang, Y. Chu, W. Shen, Z. Dou, Effect of 5-azacytidine induction duration on differentiation of human first-trimester fetal mesenchymal stem cells towards cardiomyocyte-like cells, Interact. Cardiovasc. Thorac. Surg. 9 (2009) 943–946. [8] M.-H. Kim, M. Kino-oka, M. Taya, Designing culture surfaces based on cell anchoring mechanisms to regulate cell morphologies and functions, Biotechnol. Adv. 28 (2010) 7–16. [9] M.-H. Kim, M. Kino-oka, N. Maruyama, A. Saito, Y. Sawa, M. Taya, Cardiomyogenic induction of human mesenchymal stem cells by altered Rho family GTPase expression on dendrimer-immobilized surface with d-glucose display, Biomaterials 31 (2010) 7666–7677.
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