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Normal and leukemic blood cell progenitors depend upon the bone marrow (BM) stroma with which they communicate through soluble and membrane-anchored mediators, adhesive interactions and gap junctions (GJ). Regarding hematopoiesis, it is believed that it can be influenced by connexin expression, but the exact role of GJ in cell death and proliferation is not clear. Using flow cytometry, we monitored the division rate of leukemic cell lines, communicating and not communicating with stromal cell line through GJ. We found that GJ-coupled cells (i) did not proliferate; (ii) were kept in G0; and (iii) were protected from drug-induced apoptosis when compared to either total or uncoupled cell population. We conclude that GJ coupling between stroma and leukemic lymphoblasts prevents proliferation, keeping cells in a quiescent state, thus increasing their resistance to antimitotic drugs. Since GJ are particularly abundant in the sub-endosteal environment, which harbors blood stem cells, we also asked which cells within the normal human BM communicate with the stroma. Using a primary BM stroma cell culture, our results show that 80% of CD34 þ progenitors communicate through GJ. We propose that blood cell progenitors might be retained in the low-cycling state by GJ-mediated communication with the hematopoietic stroma.

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza’s Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology.