Microscale technologies for regulating human stem cell differentiation

Abstract

During development and regeneration, tissues emerge from coordinated sequences of stem cell renewal, specialization, and assembly that are orchestrated by cascades of regulatory factors. This complex in vivo milieu, while necessary to fully recapitulate biology and to properly engineer progenitor cells, is difficult to replicate in vitro. We are just starting to fully realize the importance of the entire context of cell microenvironment-the other cells, three-dimensional matrix, molecular and physical signals. Bioengineered environments that combine tissue-specific transport and signaling are critical to study cellular responses at biologically relevant scales and in settings predictive of human condition. We therefore developed microbioreactors that couple the application of fast dynamic changes in environmental signals with versatile, high-throughput operation and imaging capability. Our base device is a microfluidic platform with an array of microwells containing cells or tissue constructs that are exposed to stable concentration gradients. Mathematical modeling of flow and mass transport can predict the shape of these gradients and the kinetic changes in local concentrations. A single platform, the size of a microscope slide, contains up to 120 biological samples. As an example of application, we describe studies of cell fate specification and mesodermal lineage commitment in human embryonic stem cells and induced pluripotent stem cells. The embryoid bodies formed from these cells were subjected to single and multiple concentration gradients of Wnt3a, Activin A, bone morphogenic protein 4 (BMP4), and their inhibitors, and the gene expression profiles were correlated to the concentration gradients of morphogens to identify the exact conditions for mesodermal differentiation.

Keywords: Human stem cells; cardiac differentiation; flow; gradients; microscale platforms; transport.

Figure 1

Mathematical modeling of flow profiles. A model microwell was designed and different flow regimes and geometries of the inlets and outlets were tested for the cells adhering to the bottom surface (numerical simulations by Comsol Multiphysics). The heat map represents the dimensionless velocity of fluids with respect to the inflow velocity. Streamlines allow visualization of the patterns followed by fluids. (a) Perfectly laminar and orderly flow with low levels of shear stress experienced by the cells generated at low flow velocities. (b) Higher velocities lead to increases in shear stress and the formation of stagnant areas within the culture chamber. (c) Poorly positioned inlets and outlets result in the formation of stagnant areas even at the proper flow rates. (A color version of this figure is available in the online journal.)

Figure 2

Microscale platform for studies of human stem cells. The platform was designed using a 3D CAD software with the following design criteria: (i) Capability of generating multiple concentration gradients in a no-shear environment, (ii) 3D culture of EBs, (iii) high-throughput, (iv) compatibility with online imaging, (v) easy retrieval of the EBs for additional analyses. The platform comprises a matrix of conical microwells accommodating EBs while shielding them from shear forces. The integrated microfluidic platform generates concentration gradients across the rows of microwells. The overall dimensions of the device are comparable to a standard microscope slide. Multiple prototypes with varying numbers of microwells were made; a prototype with five microwells per row is shown. (a) From the CAD file a mold was produced via Stereolithography Rapid Prototyping. (b) The mold could be used indefinitely to produce replicates of the microbioreactor via replica molding in poly(dimethylsiloxane). (c) A dye-tracer fluid fills the device, for visualization of the channels and microwells. (A color version of this figure is available in the online journal.)

Figure 3

Mathematical modeling of flow and mass transport. (a) Color heat map correlates to the dimensionless concentration of metabolites relative to the initial values. The concentration gradient is established from left to right across the rows of microwells. Parallel rows are replicates of the same conditions. (b) Color heat map represents the velocity. The position of two lines for further analysis is highlighted. (c) The parabolic velocity profiles across the two flow channels (left) and above the microwells (right). (A color version of this figure is available in the online journal.)

Figure 4

The formation and seeding of EBs made of hESCs and iPSCs. (a, b) Uniformly sized EBs were formed in using Aggrewell^® plates. Images refer to hESC-derived EBs. Magnifications: 4× and 10×, respectively. (c) An entire matrix composed by five rows of microwells filled with individual EBs can be visualized. Panel d: two magnifications of selected microwells. Again, a single EB fills each microwell and settles in a shear-protected environment. (A color version of this figure is available in the online journal.)

Figure 5

Expression of mesodermal genes after exposure to multiple, opposing gradients of ActivinA/BMP4 and SB-431542 or Dkk1. We quantified the expression levels of representative mesodermal and mesendodermal genes via qPCR. Data were obtained from ΔCt values and normalized to GAPDH. For each assay, 3–5 EBs per condition were pooled, from microwells in one row of the platform, corresponding to one single condition. Experiments without opposing inhibitors served as controls. hiPSC-derived EBs were subjected to a stable gradient of the chosen morphogens for 24 h between day 3 and day 4 after induction. Left inlet: Activin A (9 ng/ mL), BMP4 (13 ng/mL). Right inlet: SB-431542 (5 μM) for results in the left box, and Dkk1 (150 ng/mL) for results in the right box. Statistics were measured between control experiment and the corresponding inhibitor-based gradient for each microwell. *p <0.05, **p <0.005

Figure 6

Dye tracer study of EBs subjected to opposing concentration gradients. The microbioreactor was used to generate the opposing gradients of DAPI (left to right) and Calcein (right to left) using fluorescent tracers. hESC-derived EBs stained consistently with the dye concentrations in the double opposing gradient. (A color version of this figure is available in the online journal.)