PCTC Hematology Meeting Summary - April 29, 2019

The PCTC Hematology Meeting was separated into 4 major sessions:

   1. Reprogramming Technologies

   2. HSC Development

   3. Translational

   4. Microenvironment

George Daley presented a summary of his work to drive iPSC to become blood stem cells. The use of polycistronic vectors was a great help and episomes work well. There is thymus engraftment. There was a big point made about engraftment assays. The primary transplant assay is difficult to gauge since the cells from any experiment would be heterogeneous. A true stem cell would persist in secondaries and the Limit Dilution Assay would be accurate in secondaries and could be compared to Cord Blood. Dr. Daley also presented on mouse single cell data on the AGM and a new program called Cell Communicator that listed ligand receptor pairs that were co-regulated.

Shahin Rafii presented IPSC-derived endothelium and brain-specific endothelium. CXCL12A and BMP signaling helps the transition to blood stem cells. The polycistronic vector is more efficient. At week 3, there are hematopoietic markers, but these are immature. The morphology is like cord blood. A trick using an immune mismatch system was tried based on the increase in T cells that was expected. This showed spleen engraftment and the stem cells were functional. But the system was a little cumbersome. The other topic was on the blood brain barrier. There are published protocols to obtain these cells from IPSC, but the work shows that the protocol actually makes epithelial cells. More work will be necessary for the BBB endothelial cells.

Igor Slukvin presented on GATA2 KO conditional lines and showed that GATA2 was critical for blood stem cell induction. ETV2 is more important for hemogenic endothelial cells. Notch favors artery fates. By looking at DLL4+ or negative cells, artery is in the DLL4+ group. There also is a Sox17-/- line that impairs artery differentiation. Primitive hematopoiesis does not depend on SOX17. ATAC seq showed that the promoter proximal part of RARA was open, and there is a decrease in CDX2. This suggests that artery fate is important for the hemogenic endothelium.

Elias Zambidis presented privately funded research to look at 3i media and constructing human naïve stem cell lines. These lines have augmented differentiation. He then examined the function GFP-versions of these lines by placing them in morulas and blastulas of mouse embryos. There was extensive chimerism throughout the embryo that developed and there was extraembryonic differentiation. The hope would be to examine the AGM and fetal liver for human hematopoiesis.

Hanna Mikkola discussed her excellent work on MLLT3, which is involved in transcriptional elongation. Overexpression of MLLT3 gave HSC function and restored levels of the cells to that of a cord blood. There was a 12.5X expansion. The MLLT3 binds to the transcriptional start site and works on an elongation mechanism. MECOM and HLF are targets.  It also cooperates with DOT1l.

Nancy Speck presented excellent work on single cell RNA seq from the developing AGM. She can find specific genes for hemogenic endothelial cells before runx1 is expressed. Cell cycle genes correlate to cluster formation. She also sees that CD44 is expressed by pre endothelial cells. There also is an EMP population that is expressing specific markers including a yolk sac lymphoid population.

Trista North discussed her work on glucose increasing HSC. She found that NLRP3 is an inflammatory mediator involved in stem cell birth. This requires NFKB signaling. Pycard knockouts have less HSCs supporting her model that the inflammasome is critical to starting HSC development. She is a reporter for NFKB activity that is very helpful. Alum give more macrophages and binds to the NLRP3 receptor. Nigericin also stimulates early stem cell production. Surprisingly these drugs also work on human iPSC derived hematopoietic progenitors and there is some myeloid and B cell engraftment with the drugs.

Brandon Hadland established a coculture of AGM cells with niche cells. Using FACS for EPCR vs CD61 and index sorting, 156 HSC were generated. Using Monacle, it was possible to see good HSC markers. The cell cycle distinguished the characteristics. Looking at ligand-receptor interactions, there was several molecules of interest: notch, integrins, TGFb, wnt, VLA4, VCAM and CXCL12A. With a VLA4 inhibitor in culture there were less HSCs. These were tested with knockouts and immunobilized ligands.

Stella Chou presented excellent work on producing the first transfusion-relevant iPSC derived red blood cells. This allows blood bank typing of the specific antigens that are very relevant to transfusion sickle cell anemia patients. This approach will be very helpful for patients with allosensitization.

Thorsten Schlaeger presented work on making platelets from iPSC cells. They have found new methods to make thrombocyte progenitors and then can screen for drugs that stimulate platelet formation. The new focus is to prove that these are functional. In addition, a clinical trial is planned for this year that is funded by a company called Megakaryon.

Jason Butler presented work on aging endothelial cells. MTOR can be activated for rejuvenation and NFKB deletion in endothelial cells helps with aging. Treatment with Rapamycin leads to defects in an aged hematopoietic system and all colonies are diminished. There is more DNA damage. An Endothelial KO of MTOR showed decreased engraftment. TSP1 is expressed and the KO mice have enhanced hematopoietic recovery with increased colony number. There is no myeloid bias and good engraftment.

Leonard Zon presented work on creating a new synthetic niche for blood stem cells. He used RNA Tomography to find gene expression specific to the stem cell niche. His group found a set of endothelial genes that were specific to venous sinusoids. Making stable transgenics with the promoters driving GFP allowed for sorting of these sinusoids. ATAC seq unveiled a set of three transcription factor binding sites required for enhancer activity. Transcription factors that bind to these motifs were used in reprogramming experiments in whole embryos. These embryos developed ectopic venous sinusoids and surprisingly the stem cells traveled to these new ectopic sites. The gene expression signature is conserved in the adult kidney marrow and are found in mouse fetal liver and bone marrow. These cells may be able to be used for the treatment of myelofibrosis.

Irv Bernstein discussed clinical work with Notch ligand. 160 patients were treated and this was safe and well tolerated. The study did not demonstrate a statistically significant or clinically relevant benefit with a primary endpoint ANC500. These cells are unmatched and off the shelf. There was no difference in GVHD, but the study remains open. Cord blood is so improved and better matching so there is now a low incidence of GVHD. There is rapid CD14+ monocytes engraftment.  There is a Phase 2 study in AML patients with 220 patients.

Peter Kure discussed AML extracellular vesicles (EV). These EV suppress progenitor colony formation in vivo. There is a microRNA cargo with MiR150 and Mir155. A proteomics screen revealed issues with ribosome biogenesis.

Discussion

We need:

More introduction to the VC and Finance world to understand how the research can be supported in this early phase to promote commercialization. We have a session in September for the PCTC meeting on this.

Better humanized mouse models for testing progenitors.

More information about organoids.

Grants on the niche would be very helpful.

Support fetal research

We had a long discussion about the fact that the PCBC ended too quickly when progress was high, and there is still a need to support the basic science of progenitors and the niche.

There was a discussion about secondary transplants to assay cells produced by iPSC. This seems to be more accurate reflection on an induced cell type.

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