Answer to Question #180339 in Cell Biology for Water

Question

Mr. Guhan is a Ph.D. student studying the gene expression profile of neutrophils from blood cancer patients. He is planning to produce neutrophils from hematopoietic stem cells rather than isolating patient blood samples. But, he has no idea how to make neutrophils from stem cells. Can you give a conceptual approach to make neutrophils from stem cells? Explain how you can convince Mr. Guhan that your idea works better? What are the distinct stages of cell differentiation that occur before reaching neutrophils? Explain at least one of the major ligands and its pathways involved in the growth cycle. What are the different ways he can confirm the cells that he produced are neutrophils?

Answer 1

Functioning neutrophils are a vital component of the defense system against infection in humans.Neutrophils were generated from iPSC using embryoid body (EB) based differentiation protocols modified from Lachman et al. 18. In brief, 6 wells of confluent iPSC cultured on Matrigel were cultured in EB medium (DMEM/F12, 20% FBS, 1% NEAA) for 3 days. After 3 days, cells were washed and incubated with dispase for 5 minutes, then scored with an 18 gauge needle and scraped from the culture dish and placed in 3 wells of an ultra‐low attachment plate (Corning?) in EB medium. After 4 days of culture, 15–20 EBs (starting from 1 to 2 × 10⁶ undifferentiated iPSC) were transferred to each well of a 6‐well culture dish and cultured in StemDiff APEL 2 culture media (Stem Cell Technologies) supplemented with 25 ng/ml IL‐3 (Peprotech) and 50 ng/ml G‐CSF (Peprotech). The EBs attached to the culture plate and formed mononuclear cell forming complexes which shed hematopoietic progenitors into the culture media. Cells including myeloid progenitors and precursors were harvested every 3 or 4 days from the culture supernatant and further differentiated to neutrophils in a culture medium consisting of RPMI medium, 10% FBS, and 100 ng/ml G‐CSF for additional 5 days (hereafter called iPSC‐derived neutrophils).

genetically corrected neutrophils derived from iPSC were able to circulate and phagocytose bacteria in a model of bacterial peritonitis. These results along with the clinical experience using myeloid progenitor derived neutrophils for the prophylaxis of sepsis in neutropenic patients provide proof‐of‐concept that these unlimited source of granulocytes from myeloid progenitors, which have the ability to circulate and target infection sites, can be used for the prophylaxis and therapy of sepsis in neutropenic patients.