Introduction of exogenous genetic material into primary stem cells is essential for studying biological function and for clinical applications. Traditional delivery methods for nucleic acids, such as electroporation, have advanced the field, but have negative effects on stem cell function and viability. We introduce nanostraw-assisted transfection as an alternative method for RNA delivery to human hematopoietic stem and progenitor cells (HSPCs). Nanostraws are hollow alumina nanotubes that can be used to deliver biomolecules to living cells. We use nanostraws to target human primary HSPCs and show efficient delivery of mRNA, short interfering RNAs (siRNAs), DNA oligonucleotides, and dextrans of sizes ranging from 6 kDa to 2,000 kDa. Nanostraw-treated cells were fully functional and viable, with no impairment in their proliferative or colony-forming capacity, and showed similar long-term engraftment potential in vivo as untreated cells. Additionally, we found that gene expression of the cells was not perturbed by nanostraw treatment, while conventional electroporation changed the expression of more than 2,000 genes. Our results show that nanostraw-mediated transfection is a gentle alternative to established gene delivery methods, and uniquely suited for nonperturbative treatment of sensitive primary stem cells.

Culture conditions in which hematopoietic stem cells (HSCs) can be expanded for clinical benefit are highly sought after. Here, we report that inhibition of the epigenetic regulator Lysine-specific histone demethylase 1A (LSD1) induces a rapid expansion of human cord blood derived CD34+ cells and promotes in vitro propagation of long-term repopulating HSCs by preventing differentiation. The phenotype and molecular characteristics of cells treated with LSD1 inhibitors were highly similar to cells treated with UM171, an agent promoting expansion of HSCs through undefined mechanisms, and currently tested in clinical trials. Strikingly, we found that LSD1 as well as other members of the LSD1 containing chromatin remodeling complex CoREST are rapidly poly-ubiquitinated and degraded upon UM171 treatment. CRISPR/Cas9 depletion of the CoREST core member, RCOR1, resulted in expansion of CD34+ cells similar to LSD1 inhibition and UM171. Taken together, LSD1 and CoREST restrict HSC expansion, and are principal targets of UM171, forming a mechanistic basis for the HSC promoting activity of UM171.

The CRISPR/Cas9 system is a versatile tool for functional genomics and forward genetic screens in mammalian cells. However, it has been challenging to deliver the CRISPR components to sensitive cell types, such as primary human hematopoietic stem and progenitor cells (HSPCs), partly due to lentiviral transduction of Cas9 being extremely inefficient in these cells. Here, to overcome these hurdles, we developed a combinatorial system using stable lentiviral delivery of single guide RNA (sgRNA) followed by transient transfection of Cas9 mRNA by electroporation in human cord blood-derived CD34+ HSPCs. We further applied an optimized sgRNA structure, that significantly improved editing efficiency in this context, and we obtained knockout levels reaching 90% for the cell surface proteins CD45 and CD44 in sgRNA transduced HSPCs. Our combinatorial CRISPR/Cas9 delivery approach had no negative influence on CD34 expression or colony forming capacity in vitro compared to non-treated HSPCs. Furthermore, gene edited HSPCs showed intact in vivo reconstitution capacity following transplantation to immunodeficient mice. Taken together, we developed a paradigm for combinatorial CRISPR/Cas9 delivery that enables efficient and traceable gene editing in primary human HSPCs, and is compatible with high functionality both in vitro and in vivo.

Targeted therapies exploiting selective vulnerabilities of malignant cells are highly desired for clinical applications. The cohesin protein complex comprises of RAD21, SMC3, SMC1A as well as a fourth subunit that consists of either STAG1 or STAG2 and is essential for proper chromosomal segregation during mitosis. STAG2 loss-of-function mutations are recurrent driver events in acute myeloid leukemia (AML) and appear relatively early during leukemogenesis. Studies in cell lines have shown that STAG2 deficient cells are uniquely vulnerable to STAG1 perturbation, and this vulnerability could thus be exploited to selectively eliminate STAG2 null AML cells. Here we show that partial perturbation of STAG1 is well tolerated by normal human hematopoietic stem cells and does not affect their functionality. By contrast, STAG1 knockdown is lethal to STAG2 null human HSCs by inducing major mitotic defects. Moreover, STAG1 knockdown induced synthetic lethality in primary human AML cells harboring a STAG2 mutation and completely abrogated leukemia progression in xenograft models. Overall, our study provides proof-of-concept demonstration of a synthetic lethal approach to selectively target primary human cancer cells with STAG2 mutations

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