Hematopoietic Stem and Progenitor Cells (HSPCs): What They Are and How to Identify Them

Written by Maria Alejandra Feliz Norberto, M.S., PhD Candidate at Albert Einstein College of Medicine

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What is the blood system, and what forms it? 

The blood system, also known as the hematopoietic system, plays a crucial role in maintaining homeostasis, immunity, and oxygen transport throughout the body. It is composed of four main components: plasma, red blood cells (RBCs), white blood cells (WBCs), and platelets.¹

• Plasma is the liquid portion of blood, making up more than half of its volume. It serves as a transport medium for nutrients, hormones, waste products, and proteins such as albumin and clotting factors.  

• Red blood cells (RBCs), or erythroid cells, carry oxygen from the lungs to the tissues and remove carbon dioxide using the oxygen-binding protein hemoglobin. 

• White blood cells (WBCs) are the body’s defense system. They fight infections, coordinate immune responses, and play key roles in inflammation and tissue repair. 

• Platelets, or thrombocytes, are cells essential for blood clotting. They rapidly respond to vascular injury and help prevent excessive bleeding.

These diverse blood components are formed and replenished throughout life through a tightly regulated, dynamic biological process called hematopoiesis. 

Figure 1: The blood system. Image adapted from Khan Academy. 

What are hematopoietic stem and progenitor cells (HSPCs)?

Hematopoietic stem and progenitor cells (HSPCs) are a specialized group of immature cells found primarily in the bone marrow that are the foundational source of all blood cell types through the process of hematopoiesis. They possess two defining properties: 1. self-renewal, the ability to generate identical daughter stem cells, and 2. multipotency, the ability to differentiate into multiple blood lineages.^2-3

HSPCs can be broadly categorized into subtypes:

• Long-term hematopoietic stem cells (LT-HSCs): These are the most primitive cells, with the capacity for lifelong self-renewal and regeneration of all blood cell lineages.

• Short-term HSCs (ST-HSCs): These have limited self-renewal and contribute to blood cell production over shorter periods.

• Multipotent progenitors (MPPs): These cells lack self-renewal but retain the ability to differentiate into various blood cell types, including erythroid (e.g., red blood cells), myeloid (e.g., granulocytes, monocytes), and lymphoid lineages (e.g., B cells and T cells). 

HSPCs are essential for embryonic development, adult blood homeostasis, and rapid immune responses during infection or injury.^4-6 Their regenerative capabilities make them a cornerstone in bone marrow transplantation and cellular therapies, particularly for patients suffering from hematological malignancies, genetic blood disorders, or immune deficiencies. Moreover, because of their plasticity and self-renewal potential, HSPCs are a key focus of regenerative medicine and stem cell research.^7-8

In both basic science and clinical settings, understanding and identifying HSPCs precisely is essential, and that begins with reliable cell surface markers and high-quality antibodies. 

Figure 2. Hematopoietic hierarchy. Image adapted from Haas et al, 2018. 

Key markers and applications used to identify HSPCs.  

Identifying hematopoietic stem and progenitor cells (HSPCs) depends on detecting specific cell surface markers that distinguish these immature cells from mature blood cells. These markers are proteins expressed on the cell membrane of HSPCs and can be used to enrich, isolate, or visualize these cells. Common techniques that rely on these markers include flow cytometry (FC), immunofluorescence (IF), and western blotting (WB).^9-10 

Flow Cytometry (FC): Flow cytometry is a powerful, high-throughput technique used to analyze the expression of surface and intracellular markers on individual cells in suspension. It is the gold standard for quantifying and sorting HSPCs based on combinations of fluorescently labeled antibodies targeting key markers, such as CD34, CD117/c-Kit, and Sca-1. This technique enables researchers to assess not only the frequency of HSPCs within a heterogeneous population, like in the bone marrow or peripheral blood, but also to isolate distinct subpopulations for downstream experiments like cell culture, transplantation, or RNA sequencing. 

Immunofluorescence (IF): Immunofluorescence allows for the visualization of HSPCs and their microenvironment within tissue sections or adherent cell cultures using antibodies conjugated to fluorescent dyes. By targeting markers such as CD34 (human) or c-Kit (mouse), IF enables researchers to investigate the spatial organization, niche interactions, and differentiation status of HSPCs. IF is particularly valuable for studying stem cell behavior within specialized niches in the bone marrow and for identifying co-localization with signaling molecules or stromal cells. 

Western Blotting (WB): Western blotting is used to confirm protein expression levels and molecular weight of HSPC-associated markers in lysed cell or tissue samples. Although WB does not offer single-cell resolution or spatial context, it is particularly useful for validating marker expression during baseline conditions, during differentiation, after cytokine stimulation, or following genetic manipulation.  

HSPC Markers in Murine Models 

In mouse studies, the strategy used to identify HSPCs is through the LSK phenotype, which means cells are:¹¹ 

• Lin− (Lineage-negative): These cells lack surface markers found on mature blood cells (e.g., CD3, B220, Gr-1, Mac-1, Ter119). 

FcZero-rAb APC Anti-Mouse CD3e Rabbit IgG Recombinant Antibody (APC-FcA65651) 

Figure 3. Mouse splenocytes surface stained with PE Anti-Mouse CD4 and APC Anti-Mouse CD3e (145-2C11) Rabbit IgG RecAb (APC-FcA65651, Clone: 145-2C11) or 0.06 ug APC Rabbit IgG Isotype Control RecAb (APC-FcA98136, Clone: 240953C9). Cells were not fixed. 

• Sca-1+ (Stem cell antigen-1 positive): A marker associated with stemness. 

• c-Kit+ (CD117 positive): A receptor tyrosine kinase important for HSPC self-renewal. 

Thus, Lin− Sca-1+ c-Kit+ (LSK) cells are considered a hallmark population containing HSPCs in the mouse bone marrow. 

To distinguish the different subsets of HSPCs, further markers need to be used. Below is a list of markers used to distinguish the various subsets:¹²

• CD150+, CD48−, CD34− for long-term HSCs (LT-HSCs). 

Anti-Mouse SLAM/CD150 Rabbit Recombinant Antibody (98348-1-RR) 

Figure 4. Mouse splenocytes were surface stained with Anti-Mouse Slamf1 Rabbit RecAb (98348-1-RR, Clone: 242631G1) or 0.25 ug Rabbit IgG Isotype Control RecAb (98136-1-RR, Clone: 240953C9), and PE-Conjugated Goat Anti-Rabbit IgG(H+L). Cells were then stained with CoraLite® Plus 647 Anti-Mouse CD45R (B220) (RA3-6B2) (CL647-65139, Clone: RA3-6B2). Cells were not fixed. 

• CD34+, FLT3+ for short-term HSCs and multipotent progenitors (MPPs). 

• CD90.2 (Thy1.2) is also used in certain strain-specific gating strategies to enrich for HSCs. 

These combinations allow for the isolation of functionally distinct subsets of HSPCs and are essential for studies involving stem cell function, lineage tracing, or transplantation. 

HSPC Markers in Humans 

In human hematopoietic research (such as cord blood or bone marrow transplants), a different panel of surface markers is used to identify HSPCs. The most used markers include:¹³ 

• CD34+: A well-established marker of early hematopoietic and endothelial progenitors. It is known that CD34 is widely used to isolate stem cells for transplantation. 

• CD38−: The absence of CD38 expression helps enrich for more primitive HSCs in enriched CD34+ cells. 

• CD90 (Thy-1) and CD45RA: Used to further refine subsets of human HSCs and MPPs. 

• CD117 (c-Kit): c-Kit is expressed on early hematopoietic progenitors and is important for HSPC function. 

Anti-Human CD117/c-Kit Rabbit Recombinant Antibody (98377-1-RR)

Figure 6: (left) TF-1 cells were stained with Anti-Human C-Kit Rabbit RecAb (98377-1-RR, Clone:241313C5) and PE-Conjugated Goat Anti-Rabbit IgG(H+L)(red), or 0.25 ug Rabbit IgG Isotype Control RecAb (98136-1-RR, Clone: 240953C9) (blue). Cells were not fixed. (right) Human blood cells were stained with Anti-Human C-Kit Rabbit RecAb (98377-1-RR, Clone:241313C5) or Rabbit IgG Isotype Control RecAb (98136-1-RR, Clone: 240953C9) and PE-Conjugated Goat Anti-Rabbit IgG(H+L). Cells were then stained with CoraLite® Plus 647 Anti-Human CD34. Cells were not fixed. Lymphocytes were gated.

• CD133 (Prominin-1): Another marker used to identify primitive stem cells.

These markers enable scientists and clinicians to purify human HSPCs for research, therapy, or gene editing purposes. Functional assays such as colony-forming unit (CFU) assays or in vivo xenotransplantation into immunocompromised mice or zebrafish are often used alongside surface markers to confirm stemness and multilineage potential. 

References 

1. Handin, R. I. et al. Hematology: Basic Principles and Practice 4th edn (Elsevier, 2003). 

2. Hofer, T. et al. Fate mapping and quantitation of hematopoiesis in vivo. Annu. Rev. Immunol. 34, 449–478 (2016). 

3. Busch, K. et al. Fundamental properties of unperturbed haematopoiesis from stem cells in vivo. Nature 518, 542–546 (2015). 

4. Granick, J. L. et al. Hematopoietic stem and progenitor cells as effectors in innate immunity. Bone Marrow Res. 2012, 165107 (2012). 

5. Shevyrev, D. et al. Hematopoietic stem cells and the immune system in development and aging. Int. J. Mol. Sci. 24, 5862 (2023).  

6. Perçin, G. et al. Embryonic macrophages orchestrate niche cell homeostasis for the establishment of the definitive hematopoietic stem cell pool. Nat. Commun. 16, 4428 (2025). 

7. Bhatia, S. Long-term health impacts of hematopoietic stem cell transplantation inform recommendations for follow-up. Expert Rev. Hematol. 4, 437–454 (2011). 

8. Merli, P. et al. Hematopoietic stem cell transplantation in pediatric acute lymphoblastic leukemia. Curr. Hematol. Malig. Rep. 14, 94–105 (2019). 

9. Khan, A. et al. Flow cytometry analysis of hematopoietic stem/progenitor cells in atherosclerosis. Methods Mol. Biol. 2434, 111–123 (2022). 

10. Steidl, U. et al. Molecular biology of hematopoietic stem cells. Vitam. Horm. 66, 1–28 (2003). 

11. Ali, M. A. E. et al. Functional dissection of hematopoietic stem cell populations with a stemness-monitoring system based on NS-GFP transgene expression. Sci. Rep. 7, 11442 (2017). 

12. Purton, L. E. Adult murine hematopoietic stem cells and progenitors: an update on their identities, functions, and assays. Exp. Hematol. 116, 1–14 (2022). 

13. Henry, E. et al. Human hematopoietic stem/progenitor cells display reactive oxygen species-dependent long-term hematopoietic defects after exposure to low doses of ionizing radiations. Haematologica 105, 2044–2055 (2020).