Hydrodynamic modulation of pluripotent stem cells

Controlled expansion and differentiation of pluripotent stem cells (PSCs) using reproducible, high-throughput methods could accelerate stem cell research for clinical therapies. Hydrodynamic culture systems for PSCs are increasingly being used for high-throughput studies and scale-up purposes; however, hydrodynamic cultures expose PSCs to complex physical and chemical environments that include spatially and temporally modulated fluid shear stresses and heterogeneous mass transport. Furthermore, the effects of fluid flow on PSCs cannot easily be attributed to any single environmental parameter since the cellular processes regulating self-renewal and differentiation are interconnected and the complex physical and chemical parameters associated with fluid flow are thus difficult to independently isolate. Regardless of the challenges posed by characterizing fluid dynamic properties, hydrodynamic culture systems offer several advantages over traditional static culture, including increased mass transfer and reduced cell handling. This article discusses the challenges and opportunities of hydrodynamic culture environments for the expansion and differentiation of PSCs in microfluidic systems and larger-volume suspension bioreactors. Ultimately, an improved understanding of the effects of hydrodynamics on the self-renewal and differentiation of PSCs could yield improved bioprocessing technologies to attain scalable PSC culture strategies that will probably be requisite for the development of therapeutic and diagnostic applications.


Introduction
Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are potentially unlimited cell sources for cellular therapies due to the unique capacities of PSCs to self-renew indefi nitely and diff erentiate into cells from all three germ lineages (ectoderm, mesoderm, and endoderm) [1]. Diff erentiation of PSCs in vitro can be induced by a variety of methods, the most common of which are in an adherent monolayer format [2,3] or via formation of three-dimensional cell spheroids in suspension culture referred to as embryoid bodies (EBs) [4]. As an alternative to traditional static adherent cell culture practices that suff er from limited scalability due to surface area dependence, PSCs can be scalably expanded and diff erentiated in suspension cultures [2][3][4].
Culture systems that employ liquid motion to modulate mass transfer and shear stress, commonly referred to as hydrodynamics, include scaled-down microfl uidic systems and scaled-up bioreactor cultures. Microfl uidic systems are geometrically defi ned culture platforms that enable high-throughput screening of culture parameters, including modulation of fl uid fl ow rates, mass transfer, and shear stress. At the other end of the spectrum, bioreactors provide a potential scalable alternative to static cultures due to increased culture volumes and the ability to readily incorporate multiple sensors for bioprocess engineering strategies that facilitate continuous monitoring and feedback control. However, hydrodynamic cultures expose PSCs to physical and chemical factors not present in static culture, such as fl uid shear stress and mass transfer via convection. Th e infl uence of hydrodynamics on the self-renewal and diff erentiation of PSCs has therefore been examined in both microfl uidic and bioreactor systems. Th is review describes the current status and recent advances in understanding hydrodynamic modulation of PSCs.

Hydrodynamics
Hydrodynamics is the study of physical properties of a fl uid in motion, including velocity, pressure, density, and viscosity, as functions of space and time [5]. Mathematical solutions utilizing the conservation of mass, momentum, and energy can be obtained for theoretical models with respect to fl uid properties and system geometries. Such solutions are readily obtainable for two-dimensional adherent cell cultures, due to defi ned geometries with low fl ow rates, which enable precise characterization of fl uid fl ow in microfl uidic systems. Owing to diffi culties associated with the transfer of momentum between the Abstract Controlled expansion and diff erentiation of pluripotent stem cells (PSCs) using reproducible, high-throughput methods could accelerate stem cell research for clinical therapies. Hydrodynamic culture systems for PSCs are increasingly being used for high-throughput studies and scale-up purposes; however, hydrodynamic cultures expose PSCs to complex physical and chemical environments that include spatially and temporally modulated fl uid shear stresses and heterogeneous mass transport. Furthermore, the eff ects of fl uid fl ow on PSCs cannot easily be attributed to any single environmental parameter since the cellular processes regulating self-renewal and diff erentiation are interconnected and the complex physical and chemical parameters associated with fl uid fl ow are thus diffi cult to independently isolate. Regardless of the challenges posed by characterizing fl uid dynamic properties, hydrodynamic culture systems off er several advantages over traditional static culture, including increased mass transfer and reduced cell handling. This article discusses the challenges and opportunities of hydrodynamic culture environments for the expansion and diff erentiation of PSCs in microfl uidic systems and larger-volume suspension bioreactors. Ultimately, an improved understanding of the eff ects of hydrodynamics on the self-renewal and diff erentiation of PSCs could yield improved bioprocessing technologies to attain scalable PSC culture strategies that will probably be requisite for the development of therapeutic and diagnostic applications.
two-phase fl ow of solid suspension cells moving within the liquid medium, extensive work has been conducted to analyze fl uid dynamics in bioreactors. Dimensionless numbers can be used to describe fl ow regimes; for example, the Reynolds number is used to describe laminar and turbulent fl ow regimes. However, important parameters, such as the mixing rate and growth factor concentrations, must be determined and similitude must be met in order to use dimensional analysis for scale-up. Experimental techniques such as particle image velocimetry have been used to characterize the three-dimensional fl uid fl ow within bioreactors [6,7]. Computational fl uid dynamics techniques can simulate fl uid fl ow to solve equations governing fl uid motion [8,9], due to the diffi culties associated with obtaining exact numerical solutions to the Navier-Stokes equations for turbulent fl ow. Th e complexities of hydrodynamic conditions, includ ing intricate geometries, and spatial and temporal variations of turbulent fl ow, create challenges for examining the specifi c eff ects of individual hydrodynamic parameters on stem cell expansion and diff erentiation.
Hydrodynamic culture systems include microfl uidic systems and bioreactors that employ external agitation (rotating wall or orbiting vessels) or internal agitation (stirred-fl ask/impeller bioreactors) ( Figure 1). Although hydrodynamic eff ects can be broadly categorized as physical and chemical, the cellular processes regulating the balance of self-renewal and diff erentiation are often interconnected and thus cannot easily be attributed to a single parameter. Additionally, diff erent culture systems exhibit complex changes in multiple parameters of the fl uid fl ow and shear stress profi les, making it diffi cult to directly compare the hydrodynamic eff ects on stem cells between diff erent bioreactors. For example, stirred fl asks create turbulent fl ows (Reynolds number >1,000) with high shear stress (τ >1 dyn/cm 2 ) whereas rotating wall vessels maintain laminar fl ows (Reynolds number <100) with low shear stress (τ <1 dyn/cm 2 ), and exact values of Reynolds number and shear stress vary with rotation speed or location in the culture system [6,10,11]. Microfl uidic systems with laminar fl ows (Reynolds number <100) and physiological shear stresses (τ <20 dyn/cm 2 ) are ideal for high-throughput screening and mechanistic studies with precise control and manipu lation of fl uid properties [12][13][14]. In contrast, bioreactor systems are generally more amenable to scale-up in bioprocessing; however, the caveat is that hydrodynamic properties become increasingly heterogeneous as the reactor volume increases. Ultimately, microfl uidic systems may provide improved understanding of important hydrodynamic culture eff ects on PSCs, which can then be translated to larger-volume bioreactors for scalable, bioprocessing applications.

Hydrodynamics at the microscale
Fluid fl ow in microfl uidic systems is driven by external pressure, mechanical pumps, or pneumatic-driven pumps. Although mixing via advection between parallel laminar fl ow streams is limited, microfl uidic systems can generate gradients via diff usion with given morphogen(s) to replicate chemical concentration profi les comparable with those experienced by cells in vivo, thereby mimicking characteristics of embryonic development. Additionally, microfl uidic systems enable more systematic characterization of heterogeneous stem cell populations via single cell analysis [15] and perturbation of cell-cell and cell-material interactions [16]. Furthermore, microfl uidic devices provide high-throughput formats to examine hydrodynamic eff ects on PSCs in a cost-eff ective manner, using fewer cells and much less reagent compared with suspension bioreactors (Figure 2).

Self-renewal
In most microfl uidic perfusion cultures, fresh medium fl ows through the system continuously, with the intent of increasing cell growth by providing nutrients and removing waste products. A microfl uidic system used for expansion of human ESCs demonstrated maintenance of the pluripotency marker TG30 (CD9) over 7 days for the range of fl ow rates (3.6 to 44.3 ml/hour) investigated [17]. However, only a narrow range of fl ow rates (20.8 and 31.3 ml/hour) exhibited rates of human ESC expansion comparable with those for static cultures. Conditions at the lowest fl ow rate (3.6 ml/hour), with a Péclet number for glucose <1, demonstrated a reduced cell expansion and altered morphology [17], suggesting that decreased fl ow rates with lower nutrient renewal and waste depletion diminish PSC expansion. In contrast, reduced expansion of cells at high fl ow rates (44. ml/hour) suggested that fl uid shear was detrimental to cell growth.
Although mass transfer and fl uid fl ow are often interrelated, a microfl uidic platform was used to compare cell growth upon removal or delivery of cell-secreted factors [14,18,19]. Mouse ESC colony growth and Nanog expression were reduced when cultured under a range of shear stresses (0.063 to 16 dyn/cm 2 ) with fresh media, but not when cultured in ESC-conditioned medium at the same shear stress values, demonstrating that expansion was mediated by convective transport of soluble factors and not simply by shear stress. However, an epiblast-like phenotype, expressing Fgf5, was increased in response to shear application [14], demonstrating that shear stress elicits phenotypic changes in mouse ESCs. Even in the presence of bone morphogenetic protein 4 and leukemia inhibitory factor, which maintain the pluripotency of mouse cells in static culture, the removal of cell-secreted factors by hydrodynamic perfusion inhibited extracellular matrix remodeling and caused mouse ESCs to spontaneously diff erentiate [19]. Furthermore, the importance of autocrine and paracrine factors for maintaining pluripotency has been demonstrated both computationally, based on a combination of a  stochastic three-dimensional Brownian dynamics simulation of ligand movement and a deterministic model of ligand-mediated signaling, and experimentally, where fl ow-dependent changes in endogenously secreted gp130activating ligands impacted heterogeneity in signaling activation of signal transducer and activator of transcription 3 [20]. Altogether, these studies demon strate that although shear stress alone can induce phenotypic changes, fl uid fl ow can also modulate the transport of cell-secreted factors, thereby altering PSC pluripotency ( Figure 2).

Diff erentiation
Shear stress is generated in vivo by blood fl ow throughout the vasculature, as well as in the lymphatic and glomerular systems, and therefore has been investigated for diff erentiation of PSCs, particularly towards hematopoietic and endothelial lineages. Before the advent of microfl uidic technologies, parallel-plate chamber systems were commonly used to examine the eff ects of controlled shear stresses on cell physiology. Applied shear stresses comparable with physiologic levels in embryonic dorsal aorta (5 dyn/cm 2 ) and in large vessels (15 dyn/cm 2 ) have demonstrated increased hematopoietic [21] and endothelial [22] diff erentiation of mouse ESCs, respectively. Additionally, the mechanisms of shear-induced PSC responses, which lead to vascular endothelial cell-specifi c markers and tight junction gene expression, was medi ated by cell surface heparan sulfate proteoglycan [23]. In addition to dependence on shear stress magnitude, shear stress induced a time-dependent and reversible increase in the expression of an arterial endothelial cell marker (ephrinB2) [24], indicating that cellular phenotypes may be dynamically altered, thereby suggesting signifi cant implications for matching of in vitro culture environmental conditions with in vivo transplantation sites for the translation of PSCs in cellular therapies. Perfusion cultures also induced increased albumin secretion and urea production in human ESC-derived hepatic cells compared with static cultures [25], demonstrating the importance of hydrodynamics for generating functional diff erentiated cells and tissues. Although these studies establish a foundation for the isolation of shear stress eff ects on diff erentiation of PSCs, the eff ects of shear stress on the diff erentiated progeny of PSCs and PSCderived tissue constructs will also be important for tissue engineering, as they will probably be present in hydrodynamic conditions created in bioreactors as well as in vivo.
Microfl uidic systems can deliver proteins and signaling molecules with precise spatial and temporal control that mimics the establishment and maintenance of concen tration gradients present within developing tissues [26]. For example, decreasing concentration gradients of Wnt3a demonstrated proportional decreases in β-catenin signaling in three regions of the microfl uidic device perpendicular to the delivery of Wnt3a, using (A375) cells expressing a Wnt/β-catenin reporter [26]. In addition, diff erent cytokine solutions (sonic hedgehog with fi broblast growth factor 8 or bone morphogenetic protein 4) diff erentiated human ESC-derived neural progenitor cells into neuronal cell body clusters and neurite bundles proportional to Sonic hedgehog concentrations in a gradient chip device [27]. Furthermore, delivery of retinoic acid using a Y-channel device design with laminar fl ow of diff erent adjacent culture media compositions resulted in hemispherical neural diff erentiation patterns within EBs [12]. Hence, concentration gradients presented by microfl uidic devices can spatially control PSC signaling and diff erentiation.
As mentioned above, microfl uidic devices are able to isolate the physical and chemical eff ects of hydrodynamic culture conditions on PSCs to further interrogate the diff erent cellular outcomes for self-renewal or directed diff erentiation of PSCs [14,18,19]. In addition, microfl uidic devices provide opportunities to explore a range of hydrodynamic parameters in a systematic manner by utilizing arrays of geometric, confi guration, and operating parameters, [13,28,29]. Th e ability of micro fl uidic systems to systematically examine the physical and chemical eff ects of hydrodynamic culture parameters provides a better understanding of the biological eff ects on PSCs for engineering of hydrodynamic microenvironments, which is diffi cult in the more complex and heterogeneous fl ow environments of bioreactor systems.

Hydrodynamics in bioprocessing
Although microfl uidic systems allow increased spatial and temporal control of fl uid shear and soluble factors, diff erentiation of PSCs in suspension does not rely on sampling small cell numbers from heterogeneous stem cell populations and is not limited by surface area, and therefore off ers several advantages for scalable diff erentiation. Consequently, scale-up using suspension bioreactors is favorable for clinical applications in which the demand for large quantities (>10 7 ) of cells are anticipated [30,31]. Hydrodynamic conditions imparted within bioreactors are intended to provide enhanced mass transfer and to minimize zones of shear stress, which may cause physiological perturbations or physical damage to cells.

Self-renewal
Stem cell expansion in suspension bioreactors is typically accomplished via seeding of PSCs on microcarriers [32,33] or by the formation of three-dimensional multicellular aggregates [32,34]. Microcarriers provide a high surface area per volume for attachment of PSCs in suspension culture; however, the substrata provided by the microcarriers can infl uence PSC attachment, growth, and pluripotency [32,35]. Expansion of human ESCs as aggregates with optimized bioprocessing parametersincluding cell inoculation density, enzymatic dissociation medium, and rotation speed -resulted in a rapid scaleup strategy that produced clinically relevant numbers of human PSCs (~2 × 10 9 cells) over a 1-month period [36]. Additionally, monitoring and independent control of multiple vessels in parallel enabled the identifi cation of important bioprocess parameters for PSC expansion, including cell inoculation density and aggregate formation [37]. Alternatively, antibody blocking of E-cadherinmediated cellular aggregation allowed the proliferation of mouse ESCs as single cells in shake-fl ask bioreactors [38]. Although expansion of PSCs using blocking antibodies may not be cost-eff ective, the use of small molecule inhibitors to similarly decrease cell aggregation may off er advantages by avoiding enzymatic passaging and limiting unwanted spontaneous diff erentiation of large multicellular aggregates, as shown by Rho-associated protein kinase inhibitor in combination with the application of heat shock to enhance cell survival and increase overall cell yield of human ESC lines [39].
Interestingly, hydrodynamic culture systems have demon strated increased maintenance of pluripotency in comparison with static cultures during diff erentiation [40]. Bioreactor-derived chondrogenic, osteogenic, and cardiomyocytic cells diff erentiated from mouse ESCs aug mented the development of teratomas upon implanta tion compared with those diff erentiated in static culture [41], illustrating the persistence of pluripotency during hydrodynamic diff erentiation. Although the mecha nism for bioreactor maintenance of self-renewal is not clear, hydrodynamics appear to support increased PSC selfrenewal compared with static culture systems, even when using standard diff erentiation protocols. Hydro dynamicmediated self-renewal may off er advan tages for the expansion of PSCs but also highlights the potential safety concerns regarding the potential tumori genecity of diff erentiated PSC populations upon transplantation.
As described previously, perfusion provides continuous renewal of nutrients and elimination of waste products as well as introducing an additional external fl uid fl ow term within bioreactors that can further modulate the fl uid shear and transport profi les. Th e number of human ESCs was increased by 70% in monolayer perfusion culture compared with static conditions [42], which indicates that the continuous supply of nutrients and growth factors from conditioned medium can signifi cantly enhance PSC expansion, thereby supporting the scalability of principles described within microfl uidic systems. In addition to the infl uence of nutrients and signaling factors, controlling dissolved oxygen in a perfused stirred tank system improved the fi nal yield of expanded human ESCs by 12-fold compared with traditional static culture [43], suggesting the importance of concurrent monitoring and control of the physiochemical environment for PSC culture.

Diff erentiation
Suspension hydrodynamic cultures have been utilized to promote PSC aggregation to form EBs and subsequent diff erentiation into each of the three germ layers. Rotary wall vessels increased the effi ciency of EB formation by threefold compared with static culture, supported diff eren tiation of human ESCs into primitive blood cells, and cartilage-like structures [44], as well as improving diff eren tiation toward cardiomyocytes over static cultures [45]. Additionally, improved homogeneity of EB morphology and size have been demonstrated in stirred [46] and rotary [47] orbital cultures, which may be factors implicated in the en hanced standardization of diff erentiation within hydro dynamic cultures.
Shear stresses can be modulated within a particular culture system by altering the rotation speed to investigate the eff ects of fl uid shear on PSC diff erentiation. Within rotary orbital shakers, changes in rotation speed varied the nominal shear stress (~0.7 to 2.5 dyn/cm 2 ) and modulated the EB size, morphology, and gene expression of mouse ESCs [47,48], suggesting that subtle changes in hydrodynamic properties can aff ect the relative proportions of diff erentiated cell phenotypes. Decreases in rotation speed (10 to 20 rpm) also decreased EB size in rotary wall vessels [49]. Additionally, changes in stirring speed in a bench-scale bioreactor demonstrated an optimal speed (65 rpm) for increased cell yields and cardiomyogenic diff erentiation [50]. However, changes in rotation speed in stirred-tank systems did not alter the effi ciencies of osteogenic and chondrogenic [51] or hemato poietic [52] diff erentiation. Th ese results suggest that modulation of hydrodynamic parameters via changes in agitation speeds within bioreactor culture systems can diff erentially alter PSC diff erentiated phenotypes.
While many studies have focused on variation of mixing parameters within a single hydrodynamic system, PSC diff erentiated phenotypes can also be modulated within diff erent bioreactor confi gurations; a spinner fl ask with glass ball impeller improved human ESC diff erentiation towards cardiac and endothelial lineages over rotary wall, rotary orbital, and paddle-impeller spinnerfl ask systems [53]. In addition, diff erentiation to ckit + or sca1 + progenitor cell populations from mouse ESCs diff ered signifi cantly between hydrodynamic environments created in spinner-fl ask or rotary wall vessels [52]. In perfusion bioreactors, human ESC aggregates ex hibited similar characteristics to cells diff erentiated in vivo at the histological as well as transcriptional levels, compared with suspension EB cultures [54], highlighting a potential in vitro model that is comparable with in vivo multi-lineage diff erentiation. Although the precise mecha nisms whereby hydrodynamic cultures modulate PSC cultures remain ill-defi ned, these studies indicate that the physical and/or chemical eff ect parameters introduced by hydro dynamic mixing in bioreactors modulate diff erentiation towards specifi c lineages.
Th e numerous factors governing PSC diff erentiation are often complex and interconnected; changes in the hydrodynamic environment therefore probably alter multiple biological parameters simultaneously. Isolating such parameters could off er a more mechanistic understanding of how PSC diff erentiation is specifi cally mediated by changes in the fl ow conditions, similar to the microfl uidic studies described above. For example, it is unclear whether the previously discussed changes in PSC diff erentiation are due to the hydrodynamic environment or the EB size, both of which are modulated by rotation speed. Th erefore, by maintaining uniform popula tions of size-controlled EBs in diff erent rotation speeds (45 and 65 rpm) to isolate the impact of EB size on diff erentiation, EBs exhibited increased uniformity of diff erentiation, with subtle changes in the diff erentiation toward certain lineages [55]; however, despite the modest diff erences observed when normalizing for EB size and formation, the persistence of subtle phenotype changes indicates some role for hydrodynamics in modulating PSC fate decisions.

Induced pluripotent stem cells and reprogramming
Both mouse and human somatic cells have been reprogrammed to yield pluripotent cells [56][57][58]; however, the large-scale generation of iPSCs has been limited, at least in part due to the scalable limitations of twodimensional, static cultures and the inherent ineffi ciency of most reprogramming methods. In addition to the advantages mentioned above for the utility of scalable hydrodynamic ESC cultures, the ability to rapidly reprogram and expand iPSCs off ers additional advantages, including providing autologous sources of PSCs and enabling novel types of in vitro models of complex genetic diseases [59]. Th e expansion and diff erentiation of iPSCs have therefore been explored in hydrodynamic cultures, similar to ESCs described above, such as orbital shakers and stirred fl asks [36,[60][61][62].
In addition, somatic cells have been reprogrammed directly in suspension culture conditions. Mouse embryonic fi broblasts were transduced using retroviral vectors expressing reprogramming factors (Oct4, Sox2, Klf4 and c-Myc); after 12 days, stirred suspension cultures generated 50 million alkaline phosphatase-positive cells in suspension compared with only 4 million cells in adherent cultures [63]. Th e generation of iPSCs was also increased using doxycyline inducible reprogramming in suspension cultures to encourage apoptosis of incompletely reprogrammed cells, which cannot survive in suspension [64]. Overall, these studies demonstrate that suspension cultures can facilitate reprogramming without repeated selection via passaging of adherent cells, and therefore may improve the selection of iPSCs by taking advantage of the inability of anchorage-dependent cell populations to survive in suspension culture.

Integrated bioprocessing
One potential advantage of suspension bioreactor systems is the development of integrated processes for the scalable generation of therapeutic cell populations ( Figure 2). Integrating expansion and lineage-specifi c diff er entiation has been explored in several hydrodynamic culture systems [65][66][67]. In stirred cultures, human ESCs on microcarriers demonstrated proliferation comparable with that of human ESCs in dishes followed by effi cient transition to defi nitive endoderm after exposure to soluble stimuli in the bulk medium [65]. In rotary wall cultures, expansion integrated with osteogenic diff erentiation generated cell growth and matrix formation of mouse ESCs encapsulated in alginic acid and gelatin hydrogels [66]. Integrated bioprocessing techniques exhibited utility for expansion and cryopreservation of pluripotent human ESCs, whereby the combination of cell microencapsulation with microcarrier technology improved production and storage of human ESCs with high expansion ratios (an approximately 20-fold increase in cell concentration) and high cell recovery yields (>70%) after cryopreservation [67].
Another potential use of hydrodynamic systems is for the effi cient disaggregation of compact aggregates into single cells, for applications that require subsequent culture, purifi cation, or transplantation steps. A capillary fl ow device capable of dissociating EBs was developed by exposing the ESC multicellular aggregates to diff erent fl ow velocities (3.1, 6.2 and 8.1 m/second); however, this process resulted in the death of up to 50% of the released cells [68]. Interestingly, single cells demonstrated high viability (96%) when exposed to the highest velocity (8.1 m/second), indicating that the loss of viability is related to the dissociation of cellular adhesions rather than shear stress-mediated (25, 50 and 65 N/m 2 ) apoptosis. Using principles of fl uid fl ow to dissociate single cells from cellular aggregates or microcarriers could provide a higher throughput and less cytotoxic method than enzymatic dissociation techniques. Ultimately, integrating techniques for the expansion, diff erentiation, and cryopreservation of PSCs could increase automation and effi ciency for future bioprocessing applications.
To develop culture systems for good manufacturing practice (GMP) bioprocessing, the use of serum-free medium and automated, controlled systems via hydro dynamic bioreactors could improve the clinical trans lation of PSCs. Th e inclusion of serum in culture media creates challenges for PSC expansion and diff erentiation by introducing lot-to-lot variability and xenogenic antigens into the cultures [69]. However, serum can buff er mammalian cells from physical damage due to mechanical stresses created within bioreactor cultures [70,71]; stem cell culture in serum-free conditions could therefore make the cells more sensitive to hydrodynamic forces. Th e ability to engineer hydrodynamic culture platforms without serum was demonstrated by adjusting the medium viscosities (0.9, 40, and 70 centipoise) using carboxymethyl cellulose; overall, more homogeneous size-controlled aggregates were generated using medium with a viscosity of 40 centipoise and an optimized rotation speed (50 rpm) [36]. Th e diff erentiation of mouse ESCs in serum-free cultures containing osteogenic cell-seeded microcarriers yielded successful incorpora tion into mouse (burr-hole) fractures in the tibiae without incidence of tumor formation [33,51].
A scalable, GMP platform produced 20% myosin heavy chain and α-actinin-expressing cardiomyocytes from human ESC [72], demonstrating effi cient, scalable diff erentia tion using GMP conditions. Cryopreserved human ESC banks created under GMP conditions in stirred cultures were optimized to increase the cell expansion rate, pluri potency, and cell yields using defi ned serumfree media, seeding density, and cell splitting interval [73]. Further more, mouse ESCs expressing Oct-4, Nanog, and SSEA-1 expanded by 85 ± 15-fold over 11 days in a fully controlled stirred-tank bioreactor by fi rst optimizing the feeding regimen and cell inoculation procedure using spinner fl asks [74], indicating the ability to signifi cantly scale-up PSC expansion from laboratory-scale hydrodynamic culture systems.
Th e integration of defi ned GMP protocols within hydrodynamic cultures may provide new opportunities for PSC expansion and diff erentiation by removing the variability related to common laboratory culture procedures, such as the use of serum and frequency of manual cell handling.

Conclusions: coupling pluripotent stem cell culture and hydrodynamics
Dimensionless analysis and determination of the critical process parameters for each bioreactor system may direct PSC culture requirements; however, such parameters are expected to be diff erent between bioreactors of diff erent geometries as well as the desired cell phenotype. Additionally, the cellular processes regulating selfrenewal and diff erentiation cannot easily be attributed to a single parameter within hydrodynamic cultures. Th e behavior of hydrodynamic systems will therefore be better understood when fl uid fl ow and cell culture charac terizations can be coupled in devices to assess their interdependent infl uence in response to system perturbations. Decoupling hydrodynamic eff ects, including physical and chemical eff ects, from other perturbations in the microenvironment in high-throughput micro fl uidic systems could provide an improved understanding of the balance between the expansion and diff erentiation of PSCs, which can be translated to bioreactors for scalable, bioprocessing applications. Understanding the eff ects of hydrodynamics on pluripotent biology will enable the development of a complete bioprocess in scalable bioreactor systems for the expansion, diff erentiation, and subsequent storage of PSCs prior to their fi nal intended use. Ultimately, controlled hydrodynamic processes for the high-throughput generation of cells will minimize labor-intensive multi-step approaches for applications of PSCs in cellular therapies and tissue engineering.

Competing interests
The authors declare that they have no competing interests.