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How to Choose the Right CD34+ Cell Source for Your Research

Scientist on September 5, 2023
in Discovery & Innovation

This blog post was written by STEMCELL Technologies, who provide cell isolation products, specialized cell culture media, primary cells, and supporting reagents for use in life sciences research across the basic to translational research continuum. STEMCELL Technologies’ in-house Contract Assay Services (CAS) work with you to design and perform in vitro potency and safety studies for drug discovery and development.

What are Hematopoietic Stem and Progenitor Cells?

Due to their long research history, hematopoietic stem cells (HSCs) are among the most well-characterized stem cell populations.1 HSCs make up a small population of the hematopoietic system but have the potential to give rise to all mature blood and immune cell types. Short-term repopulating progenitor cells are also a part of the hematopoietic system, and these give rise to lineage-specific cell types. Collectively, these cells are referred to as hematopoietic stem and progenitor cells (HSPCs).

HSCs are defined by their ability to self-renew and reconstitute the hematopoietic system following transplantation, a feature that forms the basis of in vivo assays investigating HSC function. In experimental settings, the ability of human HSCs to reconstitute hematopoiesis is measured by transplantation into genetically immunocompromised mice, such as NOD-SCID-Gamma (NSG) mice, and measuring the presence of human blood cells in the blood or bone marrow (BM) after extended periods of engraftment (20 weeks or longer). In vivo engraftment is to date the best assay to measure HSC function, but it is expensive and impractical for routine use in a clinical laboratory. Therefore, in vitro assays are often used as an alternative.

The colony-forming unit (CFU) assay was first described in 1979 and is the most widely used in vitro assay for HSPCs.2 The CFU assay assesses the proliferation and differentiation pattern of hematopoietic progenitors by evaluating their ability to form colonies in a semi-solid methylcellulose medium, such as MethoCult™, supplemented with appropriate cytokines (see Figure 1). STEMCELL Technologies offers a range of cytokines and growth factors for workflows such as this — request your sample here. The CFU content of cell products has been shown to correlate with successful engraftment of HSPCs.3 Colonies derived from different types of progenitor cells are classified and counted based on morphological and phenotypic criteria. The CFU assay is widely used to study the effects of stimulatory and inhibitory growth factors, screening novel compounds to predict potential toxicity to the hematopoietic system and to test the effects of various in vitro manipulations (e.g. cell processing, cryopreservation, gene transduction, and transmission) on cellular products used in hematopoietic cell transplantation. Visit STEMCELL’s drug discovery and toxicity testing learning center to learn more.

You can learn more about hematopoiesis, the assays used to identify and quantify human HSPCs and how to identify and count colonies within the CFU assay with STEMCELL’s on-demand CFU assay training course.

What is CD34?

Cell markers, also known as surface antigens, are molecules located on a cell’s membrane used to identify specific cell types, their lineage and their stage in the differentiation process according to the presence or absence of their expression. One example is the CD34 transmembrane phosphoglycoprotein that was first identified on HSPCs, and its expression has been detected in hematopoietic and vascular tissue.

CD34 is one of the most commonly used cell surface markers to identify human HSPCs, which is expressed on HSCs, multipotent cells and more differentiated progenitor cells of individual blood cell lineages. Additional markers can be used to distinguish HSPC subsets within the CD34+ population and isolate HSPCs with different engraftment abilities and capacities to expand or generate mature blood cells in culture. Learn more about human HSPC phenotypes, frequencies and hierarchies with this wallchart. Figure 1 shows images of CFU colonies of HSPCs expressing different levels of the CD34 protein. These images were taken with STEMvision™ — an instrument designed for automating and standardizing the CFU assay.

Figure 1. CFU Frequency of Sorted CD34+ Cells. CD34+ cord blood (CB) cells were cultured in StemSpan™-XF media with CD34+ Expansion Supplement and UM171 for 7 days, before being stained with an antibody against CD34 and were bulk- and single-cell sorted for CD34high , CD34med , and CD34negative cells into the CFU assay. (A) Representative images of CFU colonies generated from bulk-sorted cells. (B) CFU frequency of each single-cell sorted population with different levels of CD34 expression. GEMM: granulocyte, erythrocyte, monocyte, megakaryocyte, BFU-E: burst forming unit-erythroid, CFU-GM: colony-forming unit-granulocyte-macrophage, CFU-M: colony-forming unit-megakaryote, CFU-G: colony-forming unit-granulocyte.

STEMCELL Technologies provides a range of sources for researchers to obtain CD34+ cells, including fresh or frozen leukopaks. Our portfolio also includes a comprehensive range of products for cell sourcing and isolation, expansion, and differentiation and analysis of HSPCs. To help ensure standardization throughout your HSPC research, use STEMCELL products from the beginning to the end of your workflow (Figure 2).

Figure 2. An Example of a Product Workflow for Hematopoietic Cell and Gene Therapy Research. Start with a reliable source of HSPCs by using fresh or frozen human blood products, including MNCs and leukopaks. CD34+ cells can be isolated from these sources using immunomagnetic EasySep™ cell isolation kits. Alternatively, start with ready-to-use human primary CD34+ cells. Human CD34+ cells can be expanded or differentiated in serum-free conditions with StemSpan™ media and supplements — such as cGMP, animal origin-free StemSpan™-AOF medium — or if working with pluripotent stem cells, with lineage-specific STEMdiff™ kits. Primary cell-derived CD34+ cells may be efficiently gene-edited using the ArciTect™ CRISPR-Cas9 System. Unmodified and gene-edited CD34+ cells can be cultured in MethoCult™ media and analyzed using the STEMvision™ instrument, now available with a software add-on for use in high-compliance environments. Perform analysis with our broad portfolio of antibodies for flow cytometry, to identify HSPC populations present after culture or measure engraftment following transplantation into immunocompromised mice when performing in vivo engraftment assays. BM MNCs: bone marrow-derived mononuclear cells, PB MNCs: peripheral blood-derived mononuclear cells.

What are the Tissue Sources of HSPCs?

HSPCs can be obtained from a variety of sources, including CB, BM and peripheral blood (PB). BM is the major site of hematopoiesis in humans after birth, and very few HSPCs are found in the PB. However, HSPCs can be mobilized from the BM and released into the peripheral blood after the injection of cytokines (specifically granulocyte colony-stimulating factor (G-CSF) and/or other agents). To this end, donors are mobilized following specific regimens with the following mobilizing agents:

  • G-CSF - The cytokine G-CSF is the most commonly used mobilizing agent and a gold standard in the clinic
  • Plerixafor - The bicyclam molecule plerixafor is a rapid-acting mobilization agent
  • Combination - The combination of G-CSF and plerixafor works synergistically, increasing CD34+ mobilization compared to single-agent mobilization

The mobilized PB (mPB) HSPCs can be collected by apheresis and are the primary source of HSPCs for clinical transplantation to treat hematologic malignancies and other blood cell disorders. Umbilical CB is also used as an alternative source of allogeneic HSPCs for patients where a suitable BM or mPB donor is not available. BM, mPB and CB are also commonly used for basic research into HSC biology and the development of novel cellular therapy applications, e.g., HSC expansion and gene therapy.

Table 1 outlines some of the advantages and disadvantages of the various sources of CD34+ cells. The most appropriate choice for your research will depend on a variety of factors, such as your application, research goals, and of course, the availability of cells from each source.

Table 1. CD34+ Cell Sources and Their Respective Features

For more frequently asked questions and answers on leukopaks, CB and BM, or to learn more about STEMCELL’s primary cell offering, read our primary cells FAQs.

Who Are STEMCELL Technologies?

STEMCELL Technologies supports pharmaceutical, biotechnology, government and academic life science organizations globally with over 2,500 specialized products and services. Our ethically sourced human primary cell portfolio includes products from fresh or frozen PB, CB and BM. We offer a range of products to help you thaw cells with confidence. Maximize post-thaw cell recovery and viability with ready-to-use CryoStor® CS10 medium. Automated cell thawing systems eliminate the risk of contamination and deliver consistent thawing profiles — use ThawSTAR® CFT2 for cryogenic vials and ThawSTAR® CB for large-volume cryobags.

Specializing in primary cell-based assays, our in-house Contract Assay Services (CAS) works with you to design and perform your drug development studies. Get the data you require by choosing from our portfolio of standardized assays or discuss your customized needs with our scientific experts.

References
  1. Weissman L. I. Stem Cells: Units of Development, Review Units of Regeneration, and Units in Evolution. Cell Press. Published January 7, 2020. Accessed August 14, 2023. https://www.cell.com/cell/pdf/S0092-8674(00)81692-X.pdf
  2. Metcalf et al.J Cell Physiology. Published February 1979.Accessed August 23, 2023. https://pubmed.ncbi.nlm.nih.gov/422666/
  3. Page M. K. et al. Total Colony-Forming Units Are a Strong, Independent Predictor of Neutrophil and Platelet Engraftment After Unrelated Umbilical Cord Blood Transplantation: a Single-Center Analysis of 435 Cord Blood Transplants. Published Sept 2011. Accessed August 14, 2023. https://pubmed.ncbi.nlm.nih.gov/21277377/
cell markers stem cells