Mechanobiology Technologies for Induced Pluripotent Stem Cells

Generation of human induced pluripotent stem cells (hiPSCs) from somatic cells via transduction with the Yamanaka factors represents a highly promising strategy to produce auto- and allogenic cell sources for therapeutic strategies as well as novel models of human development and disease. This promising stem cell technology is severely limited by low reprogramming efficiency (0.001-1%), time- and skill-intensive culture to maintain pure populations, and suboptimal directed differentiation protocols. Because current techniques for hiPSC purification remain a bottleneck, there is a great need to develop unbiased, high-throughput technologies that can efficiently separate colonies of undifferentiated hiPSCs from contaminating parental cells, feeder cells, or differentiated cells while avoiding tedious manual isolation, enzymatic dissociation of hiPSC into single cells, and labeling with antibodies or other reagents.

 We have recently demonstrated significant differences in integrin expression, FA assembly and adhesive forces between undifferentiated hiPSCs and human embryonic stem cells and parental/feeder layer cells, spontaneously differentiated and directly differentiated progenitor cells  . This distinct ‘adhesive signature’ of stem cells was exploited to rapidly (<10 min) and efficiently isolate undifferentiated cells as intact colonies from fibroblasts and spontaneously differentiated cells using microfluidics (µSHEAR) (Fig. 3). Pluripotent stem cells, irrespective of source, passage number, and feeder-free matrix, were isolated in a label-free fashion and enriched to >99% purity and survival without adversely affecting the transcriptional profile, differentiation potential or karyotype of the pluripotent cells (Fig. 4). This low-cost, label-free, high-throughput strategy is applicable to mixed cell populations with differences in adhesion strength and amenable to high-throughput analyses, real-time imaging, and in-line biochemical, genetic, and cytometric processing.

 

Figure 3. Schematic of µSHEAR microfluidics

device and scale-up.

 

 

 

 

 

 

 

 

Figure 4. µSHEAR–based isolation of hiPSC from a heterogeneous reprogramming culture. (a) Heterogeneous reprogramming culture seeded into a µSHEAR device and subjected to a shear stress of 100 dynes/cm2 for 5 min. The red arrowhead indicates a hiPSC colony that is detached by flow, white arrowhead indicates non-reprogrammed/partially reprogrammed cells. (b) Left, analysis of an unpurified reprogramming culture in devices with baseline 0.65% hiPSC purity. Center, flow cytometry plot showing detached hiPSC (TRA-1-60+CMPTX+) and non-reprogrammed/partially reprogrammed cells (TRA-1-60–CMPTX+). Right, analysis of residual cells in the device after µSHEAR.