Nuclear Isolation from Cryopreserved In Vitro Derived Blood Cells

My lab studies genes and mechanisms that affect blood cell formation. We produce blood cells from human-induced pluripotent stem cells, and we want to do this more efficiently to support clinical-scale production of cell therapies that are tailored for specific patients. We use single-cell genomics modalities to identify patterns in gene expression and other factors that can help support blood cell formation.

One major challenge for in vitro blood cell production is that is expensive and inefficient. We are using genetic approaches to address this. We want to use clues from human genetics and in vivo hematopoiesis to guide the improvement that we make to the in vitro system.

We're trying to use functional genomics and single-nucleus analyses to advance the field in terms of identifying genes and signaling networks that drive the formation of the right types of blood cells in vitro. Creating high-quality multiomic data set has helped us to discover transcription factors, energy and regular machinery that drive in vitro differentiation of blood cells. We are using this data to understand human genetic data, like GUS loci, and other certain factors that are driving differentiation that we haven't been able to see before.

We're very excited to build on insights from these multimodal data sets that we've been able to generate based on the isolation of high-quality nuclei. We intend to focus our future studies on genes and developmental mechanisms that can produce blood cell types, ultimately, at clinical scale in vitro. To begin, retrieve the cryovial with cells from liquid nitrogen and thaw it in a 37 degree Celsius water bath for two minutes.

After transferring the thawed cells to a 15-milliliter tube, gradually add 10 milliliters of pre-warmed medium, and centrifuge the tube. Discard the supernatant, and resuspend the cells in one milliliter of supplemented DPBS containing FBS and calcium chloride. Next, mix one milliliter of the cell suspension with 50 microliters each of the dead cell removal cocktail and biotin selection cocktail in a five-milliliter polystyrene round bottom tube, and incubate.

After vortexing, add 100 microliters of Dextran beads to the cell suspension, and gently mix twice with the pipette. Then add 1.3 milliliters of supplemented DPBS to the mixture, and incubate with a magnet for three minutes at room temperature. Invert the magnet and tube to pour the live cell suspension into a new 15-milliliter conical tube.

Impellet the cells in a centrifuge. After removing most of the supernatant, resuspend the cells in one milliliter of DPBS containing 0.04%bovine serum albumin. To begin, obtain enriched, live iPSC-derived cell suspension in a conical tube and centrifuge it at 460 g for five minutes at four degrees Celsius.

After discarding the supernatant, add 100 microliters of freshly prepared 0.5x cold lysis buffer. Using a P100, pipette up and down 10 times to mix the cells, and incubate the tube on ice for three minutes. Next, add 500 microliters of chilled wash buffer to the lysate.

Centrifuge the tube, and discard the supernatant. After repeating the wash, resuspend the pellet in 120 microliters of chilled diluted nuclei buffer. Using a 40-micrometer cell strainer, filter the cell suspension, and add 0.4%Trypan Blue to the filtrate.

Finally, assess the quality of isolated nuclei under a microscope. Using this technique, high-quality nuclei were isolated from cryopreserved human iPSC-derived hematopoietic progenitor cells.