Screening for Intracellular Phosphorylation Cascades That Positively and Negatively Regulate the Self-Renewing Proliferation of Immature HPCs

For the purpose of studying hematological diseases involving complex lineages of cells, hematopoietic differentiation using patient-derived iPS cells has already proved to be powerful in the analysis of various diseases. However, in terms of sourcing for the expansion of its use to larger scale studies, the time-consuming and inefficient differentiation process still presents a significant obstacle. In this context, the development of a method that allows the proliferation of progenitor cells (HPCs) which maintains their ability to differentiate into multiple lineages is an important issue.

In vivo, immature multipotent hematopoietic stem cells (HSCs) reside in the bone marrow, where intracellular signals via phosphorylation cascades co-ordinately regulate the expression pattern of transcription factors (TFs), leading to fate decisions within the microenvironment between self-renewal and one-way differentiation into HPCs. In contrast, iPS cell-derived hematopoietic differentiation does not involve HSCs and the microenvironment differs from that of bona fide cells. Therefore, the signal combinations required to amplify iPS cell-derived HPCs without further differentiation should naturally differ from those observed in HSCs in vivo. Based on this assumption, we constructed a CRISPRa and CRISPRi screen for phosphorylation-related genes using iPS cell-derived differentiation to search for factors that enable long-term maintenance of multipotent HPCs.

First, Doxycycline-inducible dCas9-VPR (CRISPRa) or dCas9-KRAB-MecP2 (CRISPRi) expression cassettes were introduced into human iPS cells before, and then acted in combination with the phosphorylation-focused sgRNA libraries at the time when HPCs begin to be produced from mesodermal progenitors during their hematopoietic differentiation. Then, to identify the sgRNAs of interest with single-cell accuracy, colony formation assays were performed by isolating HPCs one by one by single-cell sorting. 2 weeks later, cells were collected only from wells that had formed CFU-Mix. Amplicon-sequences were performed using genomic DNA extracted from those cells and the sgRNAs that significantly contributed to the phenotype were identified by calculating the difference in enrichment with control sgRNAs. As a result, the CRISPRa screen identified a group of intracellular signals related with eight candidate genes known to be specifically activated in immature cells in the normal hematopoiesis. On the other hand, the results of CRISPRi showed that suppression of necroptosis-related signalling probably promotes long-term culture of HPCs in the stroma-free, artificial culture circumstances.

Our results showed the potential to obtain multipotent HPCs from iPS cells over a long period of time and in large quantities, simply by adjusting the intracellular signals without artificial adding or removing of individual genes.