Concise review:programming human pluripotent stem cells into blood

Blood disorders are treated with cell therapies including haematopoietic stem cell (HSC) transplantation as well as platelet and red blood cell transfusions. However the source of cells is entirely dependent on donors, procedures are susceptible to transfusion‐transmitted infections and serious complications can arise in recipients due to immunological incompatibility. These problems could be alleviated if it was possible to produce haematopoietic cells in vitro from an autologous and renewable cell source. The production of haematopoietic cells in the laboratory from human induced pluripotent stem cells (iPSCs) may provide a route to realize this goal but it has proven challenging to generate long‐term reconstituting HSCs. To date, the optimization of differentiation protocols has mostly relied on the manipulation of extrinsic signals to mimic the in vivo environment. We review studies that have taken an alternative approach to modulate intrinsic signals by enforced expression of transcription factors. Single and combinations of multiple transcription factors have been used in a variety of contexts to enhance the production of haematopoietic cells from human pluripotent stem cells. This programming approach, together with the recent advances in the production and use of synthetic transcription factors, holds great promise for the production of fully functional HSCs in the future

Comparative Study of Hematopoietic Differentiation between Human Embryonic Stem Cell Lines

Directed differentiation of human embryonic stem cells (hESCs) into any desired cell type has been hailed as a therapeutic promise to cure many human diseases. However, substantial roadblocks still exist for in vitro differentiation of hESCs into distinct cell types, including T lymphocytes. Here we examined the hematopoietic differentiation potential of six different hESC lines. We compare their ability to develop into CD34+ or CD34+CD45+ hematopoietic precursor populations under several differentiation conditions. Comparison of lymphoid potential of hESC derived- and fetal tissue derived-hematopoietic precursors was also made. We found diverse hematopoietic potential between hESC lines depending on the culture or passage conditions. In contrast to fetal-derived hematopoietic precursors, none of the CD34+ precursors differentiated from hESCs were able to develop further into T cells. These data underscore the difficulties in the current strategy of hESC forward differentiation and highlight distinct differences between CD34+ hematopoietic precursors generated in vitro versus in vivo

A Defined, Feeder-Free, Serum-Free System to Generate In Vitro Hematopoietic Progenitors and Differentiated Blood Cells from hESCs and hiPSCs

Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimentional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells

Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene

The subnuclear location and chromatin state of the immunoglobulin heavy-chain (IgH) locus have been implicated in the control of V(D)J recombination. V(H)-to-DJ(H) rearrangement of distal, but not proximal V(H) genes, furthermore, depends on the B-lineage commitment factor Pax5 (BSAP). Here we demonstrate that ectopic Pax5 expression from the Ikaros promoter induces proximal rather than distal V(H)–DJ(H) rearrangements in Ik(Pax5)(/+) thymocytes, thus recapitulating the loss-of-function phenotype of Pax5(–/–) pro-B cells. The phenotypic similarities of both cell types include (1) chromatin accessibility of distal V(H) genes in the absence of V(H)–DJ(H) rearrangements, (2) expression of the B-cell-specific regulator EBF, (3) central location of IgH alleles within the nucleus, and (4) physical separation of distal V(H) genes from proximal segments in an extended IgH locus. Reconstitution of Pax5 expression in Pax5(–/–) pro-B cells induced large-scale contraction and distal V(H)–DJ(H) rearrangements of the IgH locus. Hence, V(H)–DJ(H) recombination is regulated in two steps during early B-lymphopoiesis. The IgH locus is first repositioned from its default location at the nuclear periphery toward the center of the nucleus, which facilitates proximal V(H)–DJ(H) recombination. Pax5 subsequently activates locus contraction and distal V(H)–DJ(H) rearrangements in collaboration with an unknown factor that is present in pro-B cells, but absent in thymocytes

Comparative study for the IMI2-NeuroDeRisk project on microelectrode arrays to derisk drug-induced seizure liability.

In the framework of the IMI2-NeuroDeRisk consortium, three in vitro electrophysiology assays were compared to improve preclinical prediction of seizure-inducing liabilities.Two cell models, primary rat cortical neurons and human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with hiPSC-derived astrocytes were tested on two different microelectrode array (MEA) platforms, Maestro Pro (Axion Biosystems) and Multiwell-MEA-System (Multi Channel Systems), in three separate laboratories. Pentylenetetrazole (PTZ) and/or picrotoxin (PTX) were included in each plate as positive (n = 3-6 wells) and ≤0.2% DMSO was used as negative controls (n = 3-12 wells). In general, concentrations in a range of 0.1-30 μM were tested, anchored, when possible, on clinically relevant exposures (unbound Cmax) were tested. Activity thresholds for drug-induced changes were set at 20%. To evaluate sensitivity, specificity and predictivity of the cell models, seizurogenic responses were defined as changes in 4 or more endpoints. Concentration dependence trends were also considered.Neuronal activity of 33 compounds categorized as positive tool drugs, seizure-positive or seizure-negative compounds was evaluated. Acute drug effects (<60 min) were compared to baseline recordings. Time points < 15 min exhibited stronger, less variable responses to many of the test agents. For many compounds a reduction and cessation of neuronal activity was detected at higher test concentrations. There was not a single pattern of seizurogenic activity detected, even among tool compounds, likely due to different mechanisms of actions and/or off-target profiles. A post-hoc analysis focusing on changes indicative of neuronal excitation is presented.All cell models showed good sensitivity, ranging from 70 to 86%. Specificity ranged from 40 to 70%. Compared to more conventional measurements of evoked activity in hippocampal slices, these plate-based models provide higher throughput and the potential to study subacute responses. Yet, they may be limited by the random, spontaneous nature of their network activity