Cell cycle quiescence is a key feature of hematopoietic stem cells. This protocol helps to understand the behavior of quiescent human HSCs in vitro under near physiological conditions. Using this protocol, researchers can test the effect of various compounds, nutrients, or proteins that regulate the cell cycle status as well as differentiation of HSCs in a scalable manner without using animal models.
Anti-cancer drugs have varying effects on the survival of quiescent HSCs and cycling progenitors. This protocol makes it possible to find agents that spare quiescent HSCs or agents that selectively damage quiescent leukemic stem cells. Demonstrating the procedure will be Hiroshi Kobayashi, a senior research fellow from the Takubo Laboratory.
To begin, dissolve 16 milligrams per milliliter sodium palmitate, 30 milligrams per milliliter sodium oleate, and four milligrams per milliliter cholesterol in methanol in glass tubes. Store the lipid solutions at negative 30 degrees Celsius and thaw them before use. In a fresh glass tube, mix the lipid solutions to obtain the final concentration of 100 micrograms per milliliter palmitate, 100 micrograms per milliliter oleate, and 20 micrograms per milliliter cholesterol.
Evaporate the methanol by passing nitrogen gas through the lipid solution. Completely evaporate the remaining methanol by heating the glass tube in a water bath at 37 degrees Celsius. Prepare DMEM/F-12 medium with HEPES and glutamine.
Add penicillin and streptomycin sulfate for a final concentration of 50 units and 50 micrograms per milliliter respectively. The medium can be stored at four degrees Celsius for at least two months. Add 4%of BSA to DMEM/F-12 medium with HEPES and glutamine, then adjust the pH of the medium to 7.6 using sodium hydroxide solution.
Add the medium to the glass tube with the lipids. Completely dissolve the lipids by sonication. If the medium is opaque after sonication, extend the sonication time.
When the BSA and lipids are dissolved, the samples should be stored at negative 80 degrees Celsius and used within two months. Add 0.001X of insulin, transferrin, sodium selenite, and ethanol amine mixture to the DMEM/F-12 and then filter the mixed medium using a 0.22 micrometer filter. Before use, add human stem cell factor or SCF and human thrombopoietin or TPO to the culture media at a final concentration of three nanograms per milliliter each.
Transfer 200 microliters of the previously prepared culture media with cytokines to flat bottom 96-well plates. To avoid evaporation of the medium, fill all unused wells with 100 to 200 microliters of PBS. Resuspend the sorted hematopoietic stem cells or HSCs in culture media without cytokines at 60 cells per microliter.
Aliquot 600 cells into each well. Fewer than 300 cells will lead to larger technical variation and culturing more than 1, 000 cells in a single well should be avoided because of nutrient deprivation or accumulation of unfavorable cytokines or chemokines. Culture the cells in a humidified multi-gas incubator at 37 degrees Celsius in a 5%carbon dioxide and 1%oxygen atmosphere.
After seven days of culturing the purified HSCs, up to 80%of cells displayed marker CD34 positive and CD38 negative phenotypes. The total cell number depended on the cytokine concentration. Higher concentrations of SCF and TPO induced entry into the cell cycle, proliferation and differentiation.
The number of phenotypic HSCs characterized by the marker CD34 positive, CD38 negative, CD90 positive and CD45 RA negative phenotypes increased in proportion to the SCF or TPO concentrations whereas the frequency among the total cells decreased. Following three months of transplantation of cultured adult bone marrow HSCs, reconstitution can be evaluated as a function of their frequency in the peripheral blood of human CD45 positive murine, CD45 negative Ter119 negative cells. Three lineages including CD19 positive B cells, CD13 negative, CD33 positive myeloid cells, and CD3 positive T cells were reconstituted in NOG mice transplanted with either freshly thawed or cultured HSCs.
Following culture, HSCs can be subjected to gene expression profiling such as real-time PCR and RNA sequencing. Functional validation using transplantation into immune-deficient mice can also be performed. Researchers can directly compare cycling and quiescent HSCs under defined conditions by adjusting cytokine concentrations.
This will help to understand the quiescent HSC-specific self-renewal programs, stress resistance mechanisms, and metabolic properties, which are hard to test in vivo settings.
