Biphenotypic B-lymphoid/myeloid cells expressing low levels of Pax5: potential targets of BAL development.

The expression of Pax5 commits common lymphoid progenitor cells to B-lymphoid lineage differentiation. Little is known of possible variations in the levels of Pax5 expression and their influences on hematopoietic development. We have developed a retroviral transduction system that allows for the study of possible intermediate stages of this commitment by controlling the levels of Pax5 expressed in Pax5-deficient progenitors in vitro and in vivo. Retroviral transduction of Pax5-deficient pro-/pre-B cell lines with a doxycycline-inducible (TetON) form of the human Pax5 (huPax5) gene yielded cell clones that could be induced to different levels of huPax5 expression. Clones inducible to high levels developed B220(+)/CD19(+)/IgM(+) B cells, while clones with low levels differentiated to B220(+)/CD19(-)/CD11b(+)/Gr-1(-) B-lymphoid/myeloid biphenotypic cells in vitro and in vivo. Microarray analyses of genes expressed at these lower levels of huPax5 identified C/ebpα, C/ebpδ, Pu.1, Csf1r, Csf2r, and Gata-3 as myeloid-related genes selectively expressed in the pro-/pre-B cells that can develop under myeloid/lymphoid conditions to biphenotypic cells. Therefore, reduced expression of huPax5 during the induction of early lymphoid progenitors to B-lineage-committed cells can fix this cellular development at a stage that has previously been seen during embryonic development and in acute lymphoblastic lymphoma-like biphenotypic acute leukemias.

Author Correction: Repeated dose multi-drug testing using a microfluidic chip-based coculture of human liver and kidney proximal tubules equivalents.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Repeated dose multi-drug testing using a microfluidic chip-based coculture of human liver and kidney proximal tubules equivalents.

A microfluidic multi-organ chip emulates the tissue culture microenvironment, enables interconnection of organ equivalents and overcomes interspecies differences, making this technology a promising and powerful tool for preclinical drug screening. In this study, we established a microfluidic chip-based model that enabled non-contact cocultivation of liver spheroids and renal proximal tubule barriers in a connecting media circuit over 16 days. Meanwhile, a 14-day repeated-dose systemic administration of cyclosporine A (CsA) alone or in combination with rifampicin was performed. Toxicity profiles of the two different doses of CsA on different target organs could be discriminated and that concomitant treatment with rifampicin from day6 onwards decreased the CsA concentration and attenuated the toxicity compared with that after treatment with CsA for 14 consecutive days. The latter is manifested with the changes in cytotoxicity, cell viability and apoptosis, gene expression of metabolic enzymes and transporters, and noninvasive toxicity biomarkers. The on chip coculture of the liver and the proximal tubulus equivalents showed its potential as an effective and translational tool for repeated dose multi-drug toxicity screening in the preclinical stage of drug development.

Autologous induced pluripotent stem cell-derived four-organ-chip.

Microphysiological systems play a pivotal role in progressing toward a global paradigm shift in drug development. Here, we designed a four-organ-chip interconnecting miniaturized human intestine, liver, brain and kidney equivalents. All four organ models were predifferentiated from induced pluripotent stem cells from the same healthy donor and integrated into the microphysiological system. The coculture of the four autologous tissue models in one common medium deprived of tissue specific growth factors was successful over 14-days. Although there were no added growth factors present in the coculture medium, the intestine, liver and neuronal model maintained defined marker expression. Only the renal model was overgrown by coexisting cells and did not further differentiate. This model platform will pave the way for autologous coculture cross-talk assays, disease induction and subsequent drug testing.

A four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and kidney equivalents.

Systemic absorption and metabolism of drugs in the small intestine, metabolism by the liver as well as excretion by the kidney are key determinants of efficacy and safety for therapeutic candidates. However, these systemic responses of applied substances lack in most in vitro assays. In this study, a microphysiological system maintaining the functionality of four organs over 28 days in co-culture has been established at a minute but standardized microsystem scale. Preformed human intestine and skin models have been integrated into the four-organ-chip on standard cell culture inserts at a size 100,000-fold smaller than their human counterpart organs. A 3D-based spheroid, equivalent to ten liver lobules, mimics liver function. Finally, a barrier segregating the media flow through the organs from fluids excreted by the kidney has been generated by a polymeric membrane covered by a monolayer of human proximal tubule epithelial cells. A peristaltic on-chip micropump ensures pulsatile media flow interconnecting the four tissue culture compartments through microfluidic channels. A second microfluidic circuit ensures drainage of the fluid excreted through the kidney epithelial cell layer. This four-organ-chip system assures near to physiological fluid-to-tissue ratios. In-depth metabolic and gene analysis revealed the establishment of reproducible homeostasis among the co-cultures within two to four days, sustainable over at least 28 days independent of the individual human cell line or tissue donor background used for each organ equivalent. Lastly, 3D imaging two-photon microscopy visualised details of spatiotemporal segregation of the two microfluidic flows by proximal tubule epithelia. To our knowledge, this study is the first approach to establish a system for in vitro microfluidic ADME profiling and repeated dose systemic toxicity testing of drug candidates over 28 days.