Highly sensitive droplet digital PCR method for detection of residual undifferentiated cells in cardiomyocytes derived from human pluripotent stem cells

Abstract

Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are leading candidate cells as raw materials for cell therapy products, because of their capacity for pluripotent differentiation and unlimited self-renewal. hPSC-derived products have already entered the scope of clinical application. However, the assessment and control of their tumorigenicity remains to be a critical challenge. Sensitive detection of the pluripotent cellular impurities is necessary for the safety and quality control of the hPSC-derived products. In the present study, we established a sensitive assay for detection of the residual undifferentiated hiPSCs in cardiomyocytes, using droplet digital PCR (ddPCR). The ddPCR method with a probe and primers for LIN28 significantly detected as low as 0.001% undifferentiated hiPSCs in primary cardiomyocytes, which is equivalent to the ratio of a single hiPSC to 1 × 10 5 cardiomyocytes. The ddPCR also showed that LIN28 expression is extremely low in human tissues including liver, heart, pancreas, kidney, spinal cord, corneal epithelium and lung. These results suggest that the ddPCR method targeting LIN28 transcripts is highly sensitive and useful for the quality assessment of various cell therapy products derived from hPSCs.

In Vivo Generation of Post-infarct Human Cardiac Muscle by Laminin-Promoted Cardiovascular Progenitors

“…However, in agreement with our studies, these investigators also reported higher growth rates and long-term CM adhesion on an LN matrix, as we have reported previously (Rodin et al, 2014). There are other laminin-based CM differentiation protocols (LN-521 [Kuroda et al, 2015], LN-511 [Burridge et al, 2014], and LN-211 [Minami et al, 2012]). These methods have also been shown to generate CM from pluripotent stem cells with similar differentiation efficiency, though using undefined materials.…”

Ultrasensitive and rapid quantification of rare tumorigenic stem cells in hPSC-derived cardiomyocyte populations

“…S6D). This is not an unexpected result as previous literature has indicated that pluripotent genes (i.e., POU5F1, SOX2, Nanog, Klf-4, and Lin28) are weakly expressed at the RNA level in hPSC-derived cells, typically in the range of 0.01 to 1% relative to undifferentiated hPSCs (16,26). As a result, the signal from rare hPSCs is concealed by the large background generated by a substantial excess of hPSC-derived cells.…”

Dinaciclib potently suppresses MCL-1 and selectively induces the cell death in human iPS cells without affecting the viability of cardiac tissue

“…In addition, we have treated non-purified iPS-derived cardiac cells with DMSO or 50 nM dinaciclib for 24 hrs and compared LIN28 expression using qRT-PCR as an indicator for residual iPS cells. LIN28 mRNA has recently been shown to be the most sensitive marker for detecting residual iPS cells when co cultured with human primary cardiomyocytes 26 . We have found that dinaciclib treatment decreased LIN28 to only 46% to what is seen in the control ( Supplementary Figure 1 ).…”

“…LIN28 mRNA has been shown to be the most sensitive indicator for detecting residual iPS cells when co cultured with human primary cardiomyocytes. This is based on the finding that LIN28 mRNA could not be detected in primary cardiomyocytes and is highly expressed in human iPS cells when measured by qRT-PCR 26 . Since dinaciclib has been shown to cause a reduction of the phosphorylation of serine 2 of the CTD of RNA Pol II, we speculated that it might reduce the transcription of LIN28 and falsely exaggerate the dinaciclib mediated elimination of residual iPS cells.…”