Hematopoietic Stem & Progenitor Cell Culture

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.

In this Ask the Expert Session, we will discuss challenges and techniques for the culture of HSPCs, including the use of serum-free, defined media and supplements that have been developed by STEMCELL Technologies for HSPC culture applications.

This session is sponsored by STEMCELL Technologies and hosted by Dr. Bert Wognum. Dr. Wognum is the Principal Scientistfor 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.

Questions about the culture of HSPCs?

During this Ask the Expert session, Dr. Bert Wognum will be answering your questions about StemSpan™ media and supplements for serum-free HSPC expansion, along with highlighting recent research regarding the use of small molecules for HSPC expansion.

Topics of discussion may include:

  • The best cell sources for HSPCs
  • Methods to measure HSPC frequencies and properties in culture
  • When to purify HSPCs and how (or when not to) for culture
  • How to use HSPC cultures to measure the hematotoxicity of new drug candidates
  • How to exploit molecular regulators of HSPC function to drive HSPC expansion in culture
  • Methods to generate large numbers of specific blood cells (e.g. erythrocytes and platelets) by promoting HSPC expansion and lineage-specific differentiation.
  • The use of HSPCs and culture-expanded erythroid cells as cell sources for reprogramming to generate pluripotent stem cells
  • Examples of the successful translation of methods to improve outcomes after clinical HSPC transplantation

Please take advantage of the opportunity to ask our expert a question and participate in a discussion on serum-free conditions for the ex vivo expansion of human hematopoietic stem and progenitor cells.

This session is no longer taking submissions. To be notified of new Ask the Expert sessions, join our mailing list.

Question 1

What is your advice for the best way to isolate hematopoietic stem cells from cord blood and remove erythroblasts?

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 … Continued

Question 2

What culture media and cytokines do I need to use to culture hematopoietic stem 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 … Continued

Question 3

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

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 … Continued

Question 4

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.

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 … Continued

Question 5

I am seeing doubling time of our HSCs at 36 hours. I would like to decrease that but think that this time is about average. Is this what I should expect or do you have suggestions on how to improve?

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 … Continued

Question 6

What do you think is the best transfection method for HSCs.

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. Total: 0 … Continued

Question 7

I have found some published protocols on generating iPSCs from cord blood, but I am wondering if you have a recommendation on the simplest, most effective way to do this.

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 … Continued

Question 8

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?

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 … Continued