Hematopoietic Stem & Progenitor Cell Culture

Hematopoietic stem and progenitor cells (HSPCs) are very rare in cord blood (and in other cell sources such as bone marrow and mobilized peripheral blood). For many applications it is important to obtain HSPC preparations that are relatively free of mature cells.  EasySep™ column-free immunomagnetic cell isolation may be used to either deplete mature blood cells resulting in a sample enriched for untouched CD34+ cells, or to directly select for CD34+ cells resulting in a sample with a higher purity of HSPCs. This should effectively deplete erythroblasts as these do not express CD34, but do express mature blood cell markers, such as glycophorin-A. Alternatively, standard density gradient centrifugation may be combined with RosetteSep™ selection to enrich for lineage marker-negative (Lin-) cells by depleting mature blood cells and their immediate precursors, including erythroblasts. You can also combine negative and positive selection methods (i.e., RosetteSep™ pre-enrichment followed by EasySep™ positive selection of CD34+ cells) to achieve the highest possible CD34+ cell purity from whole cord blood. For more information, please read our technical bulletin on the isolation of CD34+ cells from human cord blood. Purified CD34+ cells can be used directly in culture, or purified further by fluorescence-activated cell sorting (FACS) to isolate subsets of CD34+ cells that are more highly enriched for hematopoietic stem cells or immature progenitor cells.

That depends on the goal of the experiment and the cell types you want to obtain. For all applications I would recommend using a serum-free medium, such as StemSpan™ SFEM or SFEM II, as these have been developed to optimally support HSPCs in culture. Dependent on the application, you will need to add different combinations of cytokines. For example, if you want to increase the number of CD34+ cells you need to supplement the medium with cytokines such as TPO, SCF and FLT3-ligand. You can add one or more other cytokines such as IL-3, IL-6, GM-CSF and/or G-CSF to increase cell yields, or use previously optimized StemSpan™ expansion supplements, such as CC100, CC110 or our new CD34+ expansion supplement for more consistent results. However, you need to be aware that it is not possible to expand HSPCs indefinitely without also inducing their differentiation. Some culture conditions can transiently preserve or even increase the number of functional HSPCs during several days or weeks of culture, but eventually the cells will differentiate into mature blood cells.

Adding StemRegenin 1 (SR1) or UM171 to HSPC cultures improves the expansion of primitive hematopoietic cells, as compared to cytokine-only cultures without these small molecules.  SR1 inhibits the aryl hydrocarbon receptor (AhR), which has a role in regulating HSC activity, but beyond that, little is known. For UM171 the target molecule and pathways inside the cell have not yet been identified, although we do know the mechanism is different from AhR antagonists, as there is a synergistic effect leading to higher expansion of HSCs when SR1 and UM171 are combined in culture. For more information on the combined effects of SR1, UM171 and cytokines in stimulating HSC expansion, check out our small molecules for ex vivo HSC expansion technical bulletin.

There are many important parameters when setting up and evaluating hematopoietic cultures. First is the cell source: hematopoietic cells from bone marrow, cord blood and mobilized peripheral blood may behave differently and result in different cell yields in culture. Most likely you would want to use CD34+ cells or even subsets of this population that are more highly enriched for stem cells and primitive progenitors (e.g., CD34+CD38- cells), but there may be applications in which non-purified cells or mononuclear fractions are sufficient. The cell concentration at the start of the culture is also important. At low cell concentrations (e.g. 10ˆ4 CD34+ cells per mL of culture medium) overall cell expansion (i.e., the number of cells generated per input CD34+ cell) is generally better than at high cell concentrations and there is less chance that the medium will be depleted due to overgrowth of cells in culture. However, this is very dependent on the quality and type of cells used, and on the culture conditions, specifically the properties of the medium and the types and concentrations of cytokines used. You also need to establish clear and measurable goals about the numbers and types of cells you want to generate in your cultures and what methods are needed to characterize these cells. I would monitor the cultures closely and do cell counts (of total and viable cells) regularly. You would want to use a medium that maintains high viability (> 90%) of the cells throughout the culture. StemSpan™ SFEM II is optimized for the culture of hematopoietic cells isolated from human bone marrow, cord blood and mobilized peripheral blood, and does not contain serum or cytokines, allowing you complete flexibility in defining your culture conditions. If your goal is to expand CD34+ cells you should monitor the cultures closely for changes in the % of CD34+ cells (or CD34 subsets) by flow cytometry. If it is important that the cultured hematopoietic cells retain their progenitor or even stem abilities they will need to be tested in appropriate functional assays, such as the colony-forming unit (CFU) assay, long-term culture-initiating cell (LTC-IC) assay or transplantation assays in immunodeficient mice. For more information on these assays, please read our mini-review on Hematopoietic Stem and Progenitor Cells.

The doubling time of hematopoietic cells is very dependent on the cell source, culture conditions and the stage of differentiation. CD34+ cells from cord blood tend to proliferate faster than those isolated from bone marrow. True HSCs (typically <1% of the CD34+ cells) are quiescent and will have a lag period that can last 1 or 2 days before they undergo their first division, doubling every 24 hours or so after that, in optimal culture conditions. Erythroid progenitors can double even faster. It depends on your culture conditions as to whether it is possible to decrease the doubling time of your cells. You could try adding more or different cytokines, but be aware that most culture conditions that strongly promote the proliferation of HSCs will also induce their differentiation.

Retroviral or lentiviral transduction are the established methods to introduce genes into hematopoietic stem and progenitor cells (HSPCs), and are adequate for research applications. Lentiviral transduction has become the method of choice for gene therapy applications because it results in a higher percentage of successfully transduced HSPCs and is thought to be safer.

For the best results, it is important to begin with the correct starting repopulation for reprogramming. Culture-expanded CD34+ cells or erythroblasts isolated from cord blood serve as attractive starting populations for reprogramming. By first isolating CD34+ cells using our cell isolation products, and then expanding CD34+ cells in StemSpan™ SFEM II medium and/or driving their differentiation to the erythroid lineage with StemSpan™ Expansion Supplements, you generate a convenient starting cell population for reprogramming. In addition to products for reprogramming CD34+ cells isolated from cord blood, we offer a comprehensive workflow for generating iPSCs from CD34+ and erythroid progenitors isolated from peripheral blood.

There have been several reports of clinical trials in which ex vivo expanded cord blood (CB) cells were transplanted together with a second non-manipulated CB unit. Typically the CD34+ cells are purified first and then expanded in a serum-free medium, such StemSpan™ SFEM, supplemented with cytokines and/or other agents.

Here are four examples of published studies in which clinical benefits were observed after CB expansion was used, and a short description of the method that was used in each:

1. Delaney C et al. Nature Medicine 16: 232-236, 2010 -Cultured for 16 days with recombinant Notch-ligand and cytokines
2. de Lima M et al. N Engl J Med. 367:2305-15, 2010 - Co-cultured for 14 days with mesenchymal stromal cells and cytokines
3. Horwitz ME et al. J Clin Invest 124:3121-8, 2014 - Cultured for 21 days with nicotinamide and cytokines
4. Cutler C et al. Blood 122: 3074-81, 2013 - Stimulated for 2 hours with Prostaglandin-E2

In all studies the accelerated engraftment of neutrophils (and in certain cases, platelets) was observed. The manipulated graft contributed to hematopoiesis during first weeks or months, but long-term hematopoiesis was mostly from the non-manipulated graft. It is thus possible that the culture methods only expanded short-term repopulating cells or improved their homing ability, but it is not clear if the most primitive cells with long-term repopulating ability were expanded or maintained in these cultures. Further improvements will likely come from studies examining the importance of graft composition on engraftment (e.g., the role of T lymphocytes in either promoting or inhibiting engraftment) and from the development better conditions to maintain or expand the most primitive HSCs in culture. Some of the new small molecule regulators of HSPC function, StemRegenin 1 (SR1) and UM171, are currently being tested for their potential to improve the engraftment potential of CB engraftment, but  these studies are still at an early stage.