Culturing Hematopoietic and Progenitor Stem Cells – Key Tips and Tools

By on November 10, 2015
Expert Session Summaries

We recently finished our Ask the Expert discussion on Hematopoietic Stem & Progenitor Cell Culture. During this Ask the Expert session, we discussed HSPC culture, including the selection and evaluation of media and its components, using small molecules for HSPC expansion and factors affecting cell doubling time. Other topics included research into culture methods to increase the successful engraftment of transplanted cells, generating iPSCs from cord blood and isolating hematopoietic stem cells.

The culture of hematopoietic stem and progenitor cells (HSPCs) is important to assay the quality and functional properties of HSPCs e.g., during the implementation of transplantation or other approaches to treat hematological disorders, such as leukemia. Examples of HSPC culture assays include colony-forming unit (CFU) assays in semi-solid methylcellulose-based media, such as MethoCult™, to identify and quantify HSPCs, and expansion cultures in liquid media, such as StemSpan™, to increase HSPC numbers or generate large numbers of mature blood cells. These culture methods can also used to evaluate the efficacy and toxicity of new drug candidates on hematopoiesis in vitro and to generate target cells for reprogramming to generate induced pluripotent stem cells.

This session was sponsored by STEMCELL Technologies and hosted by Dr. Bert Wognum. Dr. Wognum is the Principal Scientist for Hematopoietic & Immunopoietic Products in R&D at STEMCELL Technologies, and currently leads the development of new media and supplements for the expansion, differentiation and detection of HSPCs in culture. Dr. Wognum obtained his PhD at the University of Amsterdam in the Netherlands and worked as a postdoctoral fellow at the Terry Fox Laboratory in Vancouver, Canada, and the Department of Hematology at Erasmus University in Rotterdam, the Netherlands. He studied the role of hematopoietic growth factors and their receptors in normal hematopoiesis and leukemia and contributed to preclinical studies aimed at improving hematopoietic recovery after cytoreductive therapy and transplantation.

Below is a sneak peek of the discussion, for a full transcript, please see – Ask the Expert – Hematopoietic Stem & Progenitor Cell Culture.


We are about to begin a media evaluation for our hematopoietic cultures. We don’t have time/resources to do a completely exhaustive study, so what would you recommend we use as our key parameters for evaluation. We have 6 media that we are looking at and then will take 2 to more extensive study.

The Answer:

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.


How effective are some of the new stem cell activators, such as SR-1 and UM171? Is it known how these molecules work?

The Answer:

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.


I saw you listed in your blog – Examples of the successful translation of methods to improve outcomes after clinical HSPC transplantation. Could you elaborate? What do you feel are the important culture conditions that translate to transplantation success?

The Answer:

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.

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