Label-Free and High-Throughput Removal of Residual Undifferentiated Cells From iPSC-Derived Spinal Cord Progenitor Cells

Abstract

The transplantation of spinal cord progenitor cells (SCPCs) derived from human-induced pluripotent stem cells (iPSCs) has beneficial effects in treating spinal cord injury (SCI). However, the presence of residual undifferentiated iPSCs among their differentiated progeny poses a high risk as these cells can develop teratomas or other types of tumors post-transplantation. Despite the need to remove these residual undifferentiated iPSCs, no specific surface markers can identify them for subsequent removal. By profiling the size of SCPCs after a 10-day differentiation process, we found that the large-sized group contains significantly more cells expressing pluripotent markers. In this study, we used a sized-based, label-free separation using an inertial microfluidic-based device to remove tumor-risk cells. The device can reduce the number of undifferentiated cells from an SCPC population with high throughput (ie, >3 million cells/minute) without affecting cell viability and functions. The sorted cells were verified with immunofluorescence staining, flow cytometry analysis, and colony culture assay. We demonstrated the capabilities of our technology to reduce the percentage of OCT4-positive cells. Our technology has great potential for the "downstream processing" of cell manufacturing workflow, ensuring better quality and safety of transplanted cells.

Keywords: cellular therapy; induced pluripotent stem cells; neural stem/progenitor cell; sorting technologies; spinal cord injury.

Graphical Abstract

Figure 1.

iPSC neural differentiation results in size and nucleus area reduction. (A) Microscopy images of suspended CLEC-iPSCs and CLEC-SCPCs. (B) Size profiling of the CLEC-iPSC and CLEC-SCPC using our customized MATLAB code. Cell size was significantly different, P < .001, Mann-Whitney test (n = 1326 cells). (C) Flow cytometer analysis of the SCPCs. The large cells gated in the SSC-A/FSC-A plot were mostly OCT4-positive cells. The FSC intensity is proportional to the diameter of the cells in flow cytometry. (D) The OCT4 fluorescent intensity is correlated with the size of SCPCs (based on FSC intensity). Rank correlation by Spearman analysis (R = 0.68) indicates a moderate/good correlation between cell size and OCT4 level. The dashed line gated OCT4 positive and negative cells. (E) Fluorescent images of DAPI staining in CLEC-iPSCs, and CLEC-SCPCs differentiated for 4 and 10 days. The nucleus area of iPSCs was reduced during the differentiation processes. (F) Quantification of the nucleus area of the iPSCs, CLEC-SCPCs differentiated for 4 and 10 days. The nucleus area was reduced significantly after 4 days (P < 0.001), and after 10 days (P = 0.016), Kruskal-Wallis test was followed by Dunn post hoc test (n = 1200 cells/group).

Figure 2.

Sorting SCPCs and iPSCs using a microfluidic MDDS sorter. (A) Schematic of MDDS sorter (left) and representative bright-field image (right) showing cells were sorted into 5 outlets ranging from the smallest (S1) to the largest (S5). (B) SCPCs spiked with iPSCs were sorted using an MDDS sorter. The sorted cells were analyzed by immunofluorescence with OCT4 (red), SOX1 (green), HOXB4 (green), and DAPI (blue). (C) Quantification of cell size, OCT4, SOX1, and HOXB4 expression by unsorted group and sorted groups (ie, S2-S4). Cell size was significantly different, P < .001, Mann-Whitney test (n = 340). Significant difference in the expression of OCT4 and HOXB4 between the unsorted and S2 groups was observed. There was a significant difference in the expression of OCT4, SOX1, and HOXB4 between the S3 and S4 groups. Kruskal-Wallis test was followed by Dunn post hoc test (n = 15).

Figure 3.

Optimization of SCPC sorting using a microfluidic MDDS sorter, transitioning from a constant-speed sorting (E, F) to a low-high-speed sorting (A-D). (A) Representative bright-field images showing cell separation under low speed (ie, 2 mL/minute) and high speed (ie, 3 mL/minute), enabling the collection of smaller and larger cells, respectively. (B) Representative microscopy images of Unsorted, Small, and Large groups using low-high-speed setting. (C) Box plot graph of cell size for the Small and Large groups (with a mean difference of 2.9 µm). The cell size varies significantly among the groups (P < .001), as determined by the Kruskal-Wallis test, followed by a Dunn post hoc test (n = 324 cells/group). (D) Viability of the Unsorted, Small, and Large groups using low-high-speed sorting (n = 3). Cell size is significantly different among groups, P < .001, Kruskal-Wallis followed by a Dunn post hoc test (n = 324 cells/group). (E) Representative bright-field image showing cells sorted into Small (S2) and Large groups (S4 and S5) at a constant flow rate of 2 mL/minute. (F) Box plot graph shows the size distribution of the Unsorted, Small, and Large groups at a constant flow rate of 2 mL/minute (mean difference of 1.5 µm). (G) Schematic diagram depicting the recirculation strategy to re-sort cells for low-high-speed setting.

Figure 4.

Immunostaining and colony culture assay of sorted CLEC-SCPCs. The SCPCs were separated into Small and Large groups by the recirculation strategy. (A) Immunofluorescence images of the Unsorted group and the sorted (Small and Large) groups with OCT4, SOX1, and DAPI, along with the merged images. (B) Percentage of OCT4⁺SOX1⁻ cells in the Unsorted, Small, and Large groups (one-way ANOVA followed by the Tukey post hoc test, n = 3). (C) Comparison of cell nucleus area of the Unsorted, Small, and Large groups (Kruskal-Wallis test was followed by Dunn post hoc test, n = 5000 cells/group). (D) Colony culture assay. SCPCs and SCPCs spiked with iPSCs were cultured in an iPSC growth medium on Matrigel coating for 6 days before immunostaining and imaging. The SCPCs spiked with iPSCs were used as a positive control group to observe colony morphology formed from spiked iPSCs. Colonies were observed in phase-contrast images of both SCPCs, and SCPCs spiked with iPSCs. Two types of colony morphology were observed: defined colony (with a sharp edge, compact cells pointed by number 1) and immature colony (with a defined edge, large nucleus pointed by number 2 for SCPCs spiked iPSCs group and number 3 for SCPCs groups). These colonies were verified by immunofluorescence images with OCT4 (red), DAPI (blue), and the merged image overlaid with phase contrast. (E) The map of identified colonies based on colocalization of phase contrast and OCT4 staining maps. The map consisted of 10 × 10 images, and identified colonies were marked with white “X.” (F) The number of quantified colonies in the Unsorted, Small, and Large groups (one-way ANOVA followed by the Tukey post hoc test, n = 3).

Figure 5.

Flow cytometry assay shows that the percentage of OCT4⁺ cells is consistently lower in the sorted Small group regardless of cell culture batches and cell lines. (A) Flow cytometry analysis of the Unsorted group and the Small and Large groups from 3 batches of day 10 CLEC-SCPCs generated separately and day 10 BJ-SCPC. (B, C) The percentage of OCT4⁺ and OCT4⁺SOX1⁻ cells, respectively, of the Unsorted, Small, and Large groups. (D) The removal rate of OCT4⁺ and OCT4⁺SOX1⁻ cells by removing the Large group (method for calculating removal rate is described in Methods section).