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Introduction

Most blood cells have a finite lifespan and are replaced throughout life by the proliferation and differentiation of a very small population of pluripotent hematopoietic stem cells (HSCs). HSCs make up a very small population of the hematopoietic system, however, these cells possess the unique ability to self-renew and undergo successive stages of differentiation, ultimately giving rise to mature blood cells (Figure 1). Also making up a part of the hematopoietic system are short-term repopulating progenitor cells, which give rise to lineage-specific cell types. Collectively, these are referred to as hematopoietic stem and progenitor cells (HSPCs).

The bone marrow (BM) is the primary site of hematopoiesis in humans after birth. Certain treatments, such as cytokine administration (notably granulocyte colony-stimulating factor; G-CSF), myelosuppressive drugs used in cancer therapy, and agents disrupting hematopoietic-BM stromal cell interactions, facilitate rapid mobilization of stem and progenitor cells into circulation. Transplantation of BM or mobilized peripheral blood (mPB) from HLA-matched donors is a well-established therapy for leukemia and various hematologic and immune disorders. Moreover, umbilical cord blood (CB) obtained at birth and cryopreserved provides an alternative source of HSPCs, particularly for patients without suitable BM or mPB donors. Read more about the different sources of HSPCs here.

Recent technological advances in maintaining or amplifying HSPC populations in culture, alongside CRISPR/Cas9 gene editing techniques, have catalyzed the emergence of innovative cell and gene therapy approaches to treat genetic blood disorders like sickle cell disease and thalassemia. The development of cell and gene therapy applications depends on the progress of fundamental research into HSPC biology, predominantly explored through murine models, and the refinement of methodologies for HSPC identification, isolation, and functional characterization from diverse tissue sources, such as bone marrow, blood, and other tissues.

This review aims to provide a comprehensive overview of transplantation and culture assays devised to evaluate the hematopoietic potential of HSPCs. It further delves into phenotypic markers and methodologies for isolating mouse and human HSPCs. Additionally, the review explores culture systems tailored to expand limited HSPC pools in clinical grafts, promote HSPC proliferation and lineage-specific differentiation toward mature blood cell lineages, and generate HSPCs and blood cells from pluripotent stem cells (PSCs).

In Vivo Assays for Hematopoietic Stem and Progenitor Cells

An important characteristic of an HSC lies in its ability to restore blood cell production after transplantation. Experimental designs involving the transplantation of BM cells or purified HSPC subsets in mice have proven invaluable for understanding the biology of both mouse and human stem cells.

Transplantation Studies Using Mouse Cells

To assess the hematopoietic potential of mouse HSCs, BM cells are typically injected into mice whose hematopoiesis has been suppressed, commonly through irradiation. The subsequent repopulation of recipient mice with cells derived from the donor is monitored periodically in blood samples to examine repopulation kinetics. Furthermore, after a minimum of four months, donor-derived cells are evaluated in various tissues including BM, spleen, blood, and thymus to ascertain long-term engraftment (Szilvassy et al., 2002).

The transplantation assay format varies in the genetic backgrounds of donor and host mouse strains, the methodology employed to ablate or suppress hematopoiesis in the recipient pre-transplantation, the detection techniques utilized to identify the progeny of donor-derived stem cells, and the defined endpoints and criteria for determining "successful" engraftment.

In one type of assay, recipient mice undergo total body irradiation to eliminate HSPCs. Subsequently, these mice receive intravenous injections of BM cells or purified HSPCs from a donor mouse, along with "competitor" cells from the same mouse strain as the recipient mice (Purton et al., 2000). These competitor cells serve multiple purposes: providing short-term radioprotection, ensuring recipient survival, and exerting selective pressure to identify stem cells with robust competitive repopulating potential. Alternatively, other assays utilize recipient mice with defective endogenous hematopoiesis due to mutations in the Kit gene. In these models, transplantation with donor cells from wild-type mice is feasible without the need for co-transplanted radioprotective cells (Miller & Eaves, 1997).

The prevalent method for tracing the progeny of transplanted HSCs involves using donor and recipient mouse strains expressing different isoforms of the pan-leukocyte antigen CD45 (CD45.1 or CD45.2). This distinction enables the differentiation between donor and recipient cells via flow cytometry (Spangrude et al., 1988). While this technique effectively identifies donor-derived lymphocytes, granulocytes, monocytes, and HSPCs, it cannot detect donor-derived erythrocytes and platelets, as these cells lack CD45 expression. However, by employing transgenic donor cells expressing a readily detectable reporter molecule, such as green fluorescent protein (GFP), in all blood cell lineages, including the erythroid lineage, the engraftment of transplanted cells can be quantified across all cellular lineages (Boyer et al., 2019).

In this experimental setup, different cohorts of mice receive varying quantities of donor cells. After several months, the extent of hematopoietic reconstitution in each group of mice is evaluated. Utilizing Poisson statistics, researchers can then determine the frequency of repopulating cells within the transplanted cell population (Szilvassy et al., 1990). This methodology, complemented by single-cell transplantations wherein purified HSPCs are individually injected into groups of mice, facilitates a detailed quantitative analysis of HSC frequencies across diverse purified HSPC subsets. Moreover, it enables the investigation of engraftment dynamics and differentiation potential at the individual HSPC level. To investigate the in vivo self-renewal capacity of mouse HSCs, researchers can also perform serial transplantations, wherein bone marrow cells from primary recipients are transplanted into secondary, tertiary, and even quaternary recipients.

Recent advancements have revolutionized the study of HSCs by enabling the labeling of millions of individual cells within a heterogeneous population with unique genetic markers (“barcodes”). These markers, integrated into the genome, are inherited by subsequent generations of daughter cells. Upon transplantation of numerous tagged HSCs into recipient mice, the engraftment kinetics, size, and lineage distribution of each labeled HSC can be precisely quantified. This is achieved by identifying the specific barcode within blood cells using high-throughput parallel sequencing methods (Naik et al., 2014). This approach facilitates a comprehensive clonal analysis of multiple HSCs, overcoming the inherent limitations of traditional single-cell transplantation experiments.

Transplantation Studies Using Human Cells

The hematopoietic potential of human HSPCs can be investigated through transplantation into genetically immune-deficient mice, followed by the assessment of human blood cells in the blood, BM, or other organs of these mice several weeks to months later.

Traditionally, these assays were conducted using the severe combined immunodeficiency (SCID) and non-obese diabetic (NOD)/SCID mouse strains. However, these approaches required large cell numbers to overcome immune rejection by residual host macrophages and natural killer (NK) cells. Moreover, these assays exhibited a bias towards B-lymphocyte development, while impeding sustained long-term production of myeloid cells, platelets, and erythrocytes. Consequently, studying the kinetics of human cell engraftment or distinguishing between short-term and long-term reconstitution mediated by distinct HSC subsets was not possible.

The development of other immunodeficient mouse strains has alleviated many of these challenges. Specifically, β2-microglobulin-deficient and interleukin-2 receptor (IL-2R) γ-deficient NOD/SCID mice demonstrate robust engraftment levels for over 20 weeks post-transplantation (Cheung et al., 2013; Ito et al., 2002; Kollet et al., 2000; Shultz et al., 2005). IL-2Rγ-deficient mice, coupled with functional impairment of endogenous HSCs due to loss-of-function mutations in the Kit gene, exhibit even greater permissiveness for human HSC engraftment and prevent the need for pre-transplant conditioning via irradiation (Cosgun et al., 2014).

These enhanced xenotransplantation assays have facilitated a more comprehensive characterization of human HSCs and have been instrumental in studies identifying human HSCs at single-cell resolution (Notta et al., 2011). For further insights into the historical context of xenogeneic mouse transplantation assays and the utilization of other mouse strains, please refer to a recent review by Mian et al. (2021).

In Vitro Assays for Hematopoietic Progenitor Cells

While transplantation assays are essential for evaluating the engraftment potential and hematopoietic reconstitution ability of HSPCs in vivo, culture assays have also emerged as valuable tools for assessing the proliferative and differentiation capacities of these cells. These assays are capable of detecting hematopoietic cells at various stages of differentiation, ranging from multipotent progenitors to lineage-restricted progenitor cells. In the subsequent sections, we will delve into the principles and applications of two of the best-characterized culture assays, the colony-forming unit (CFU) assay and the long-term culture-initiating (LTC-IC) assay.

Figure 1. Hematopoietic Hierarchy, Assays and Markers in Mice and Humans

Schematic representation of the hematopoietic hierarchy from HSCs, via intermediate progenitor stages, to mature blood cells. Assays to identify HSCs and progenitors are shown at the top. The most definitive phenotypical markers used to identify the various types of mouse and human hematopoietic cells are shown at the bottom. Additional markers can be used to further distinguish between subsets. Refer to the text for further details. It should be noted that this model is continuously being revised and updated and several other ways of presenting the hematopoietic hierarchy and differentiation trajectories have been proposed (Laurenti & Gottgens, 2018). For example, HSCs are more heterogeneous with respect to self-renewal and differentiation abilities, and lineage commitment and differentiation are more continuous than suggested by surface marker expression profiles and functional criteria alone. The model also doesn't show the vast differences in the numbers of individual types of blood cells generated, with erythrocytes and neutrophils representing ~97% of the ~300 billion new blood cells (excluding platelets) produced in the human bone marrow every day under steady-state conditions. (Sender & Milo, 2021).
LT-HSC: Long-Term Hematopoietic Stem Cell; ST-HSC: Short-Term Hematopoietic Stem Cell; MPP: Multipotential Progenitor; CMP: Common Myeloid Progenitor; CLP: Common Lymphoid Progenitor; CFU-GEMM: Colony-Forming Unit – Granulocyte/Erythrocyte/Macrophage/Megakaryocyte; BFU-E: Burst-Forming Unit – Erythroid; CFU-E: Colony-Forming Unit – Erythroid; CFU-Mk: Colony-Forming Unit – Megakaryocyte; CFU-GM: Colony-Forming Unit – Granulocyte/Macrophage; CFU-G: Colony-Forming Unit –Granulocyte; CFU-M: Colony-Forming Unit – Macrophage

Colony-Forming Unit Assays

Since its inception over five decades ago (Bradley & Metcalf, 1966), the , also called colony-forming cell (CFC) assay, has emerged as the gold standard in vitro functional assay for investigating hematopoietic progenitor cells. In the CFU assay, cells are cultured at low cell density in a semi-solid medium, typically methylcellulose-based (e.g. �ѱ�ٳ�ǰ�ܱ���™), and supplemented with appropriate cytokines. These conditions support the proliferation and differentiation of individual progenitor cells, or CFUs, which give rise to discrete colonies in usually one to two weeks of culture. Distinct colonies arising from various types of progenitor cells are identified and quantified based on the number and types of mature cells they contain, utilizing morphological and phenotypic criteria. The CFU assay is primarily employed to detect multipotential and lineage-restricted progenitors of the erythroid, granulocytic, and macrophage lineages. Moreover, megakaryocyte and B-lymphoid progenitors can also be identified by applying selective culture conditions tailored to these specific cell types.

Widely utilized across research and clinical settings, the CFU assay is an important tool for examining the impacts of stimulatory and inhibitory agents on HSPC proliferation and differentiation, including potential toxicities of new drugs. Learn more in our whitepaper, The CFU Assay in Preclinical Toxicity Testing. Additionally, it facilitates the evaluation of in vitro manipulations, such as cell processing, cryopreservation, genetic modification, and ex vivo expansion culture, thereby ensuring the quality of cellular products utilized in hematopoietic cell transplantation and other therapeutic interventions.

Multiple studies have linked CFU counts to key transplantation outcomes, such as engraftment time and overall survival post-transplantation (Hogge et al., 2000; Iori et al., 2004; Page et al., 2011; Prasad et al., 2008; Yang et al., 2005; Yoo et al., 2007). Consequently, this assay is valuable for predicting graft quality, notably aiding in selecting cord blood units with viable progenitor cells before transplantation. Automated imaging platforms, such as , streamline colony counting, enhancing reproducibility and standardization (Velier et al., 2019).

Markers and Methods for HPSC Isolation

In addition to the functional assays described in the previous paragraphs, HSPCs can also be distinguished based on their phenotype. This involves staining cells, typically with fluorescently labeled antibodies targeting cell surface antigens or DNA dyes, and subsequently analyzing through flow cytometry or microscopy. This method has greatly enhanced our understanding of key markers distinguishing HSCs, progenitors, and mature blood cells. Such insights have been essential for devising strategies to isolate HSCs from bone marrow (BM), blood, and other heterogeneous cell populations.

This section will delve into the most commonly utilized markers for identifying and purifying HSCs in mice and humans. Additionally, a summary detailing additional markers utilized to differentiate progenitor subsets, including common myeloid progenitors, common lymphoid progenitors, granulocyte and macrophage progenitors, and megakaryocyte and erythroid progenitors, was previously provided in Figure 1.

Mouse Cells

The initial step in identifying and isolating mouse HSPCs in BM, spleen, fetal liver, or other tissues usually involves the elimination of mature cells that express “lineage" (Lin) antigens specific to terminally differentiated blood cells. Lin antigens, such as CD3 for T cells, B220 for B cells and NK cells, Ly6G/Gr-1 for granulocytes, CD11b/Mac-1 for monocytes and macrophages, and TER-119 for erythroid cells, are absent or expressed weakly on HSPCs.

Following the elimination of lineage-positive (Lin+) cells through immunomagnetic isolation methods like �������⳧���™, HSPCs can be further characterized using combinations of cell surface markers, notably KIT (i.e. the receptor for stem cell factor [SCF], also known as CD117) and SCA1 (Muller-Sieburg et al., 1986; Okada et al., 1992; Osawa et al., 1996; Spangrude et al., 1988; Uchida et al., 2003).

Lin-SCA1+KIT+ (LSK) cells account for less than 0.1% of nucleated BM cells but contain most repopulating HSCs. However, SCA1 is only useful in some mouse strains (e.g. C57Bl/6), but not in BALB/c and other mouse strains which exhibit low SCA1 expression on HSCs (Spangrude & Brooks, 1992, 1993). Alternatively, markers and isolation strategies based on a Lin-CD48-CD150+ phenotype (the SLAM phenotype) are effective across most mouse strains (Kiel et al., 2005).

LSK and SLAM cells represent heterogeneous populations, with HSCs accounting for at most 10 - 20% of all cells. Further enrichment of HSCs, achieving frequencies as high as 50%, has been accomplished by selecting LSK or SLAM cells expressing elevated levels of CD201 (endothelial protein C receptor; EPCR) and low or undetectable levels of CD34, CD135 (FLT3), and CD49b. These enriched HSC populations retain low levels of DNA dyes like Rhodamine-123 (Rho123) and Hoechst 33342 due to the high expression of multidrug transporter proteins MDR1 and ABCG2, respectively (Balazs et al., 2006; Benveniste et al., 2010; Kent et al., 2009; Kiel et al., 2005; Osawa et al., 1996; Uchida et al., 2003; Uchida & Weissman, 1992).

Hematopoietic Stem and Progenitor Cell Expansion Cultures

Umbilical cord blood (CB) serves as an alternative source of HSCs for transplantation in patients lacking a suitable BM or mPB donor. However, most CB units lack sufficient numbers of primitive cells to facilitate successful engraftment in adult recipients. To overcome this challenge, extensive research has focused on developing culture methods capable of augmenting the numbers and engraftment potential of repopulating cells, aiming to enhance the clinical applicability of CB for cellular therapy.

One approach to increase HSPC numbers involves culturing CD34+ cells with combinations of cytokines, such as IL-3, IL-6, SCF, Flt3L, TPO, GM-CSF, and G-CSF. This method has demonstrated a remarkable capability to expand CD34+ cell numbers by 100 to 1000-fold within 7 - 20 days of culture. However, typical culture conditions tend to induce differentiation in most HSPCs, resulting in the loss of primitive HSPCs and their replacement with more differentiated cells, which may outcompete any remaining HSPCs by the end of the culture period.

To enhance the frequency and yields of primitive HSPCs post-culture, several strategies have been devised. These include culturing CD34+ cells or highly purified primitive HSPCs at low cell densities, with frequent medium changes or in fed-batch cultures, to mitigate the impact of inhibitory factors secreted by differentiating cells (Csaszar et al., 2012). Other advancements involve the utilization of supportive mesenchymal stromal cells (de Lima et al., 2012), immobilized Notch-Ligand (Delaney et al., 2010), or the incorporation of specific soluble small molecule HSC agonists into the culture medium. Noteworthy small molecule agonists whose effectiveness has been validated include nicotinamide, StemRegenin 1 (SR1), and UM171 (or its analog, UM729) (Boitano et al., 2010; Fares et al., 2014; Peled et al., 2012; Subramaniam et al., 2020). Supplementing CD34+ cultures with small molecule agonists has been shown to elevate HSC numbers by approximately 10-fold, as determined by transplantation assays in immunodeficient mice, compared to cultures with cytokines alone.

Some of the enhanced HSPC expansion techniques have been successfully implemented in clinical trials to improve the engraftment of transplanted CB CD34+ cells in patients with hematologic malignancies. Specifically, HSPCs expanded with cytokines, along with either nicotinamide or UM171, supported earlier neutrophil and platelet recovery along with improved long-term multi-lineage engraftment compared to single unmanipulated CB transplantation (Cohen et al., 2020; Horwitz et al., 2021). The sustained engraftment of culture-expanded HSCs relied on the co-infusion of donor T cells, which likely prevent the elimination of cultured HSCs by residual patient T cells or, in the case of double CB transplantation, by donor T-cells from a second unmanipulated graft (Cohen et al., 2020; Horwitz et al., 2014; Wagner et al., 2014). These studies offer compelling evidence that ex vivo HSPC expansion can be advantageous for clinical transplantation.

Several differentiation protocols have been developed to generate hematopoietic cells from PSCs. In one approach, PSCs are plated as clumps or single cells on a layer of solubilized basement membrane matrix components (e.g. Matrigel®) to form a two-dimensional cell layer. In another method, PSCs are aggregated to form 3-dimensional embryoid bodies. In both methods, PSCs undergo initial differentiation into mesodermal cells during 3 - 4 days of culture with cytokines, such as vascular endothelial growth factor (VEGF), bone morphogenetic protein 4 (BMP4), or basic fibroblast growth factor (bFGF).Subsequently, hematopoietic growth factors such as SCF, TPO, IL-3, and IL-6 are introduced to drive the differentiation of mesodermal cells into hematopoietic progenitors. This phase of culture typically spans 7 - 9 days, following which cells are harvested. The cells are then assessed by flow cytometry to identify cells expressing hematopoietic markers, notably CD34, CD45, and CD43, and their ability to generate myeloid, erythroid, and multi-lineage colonies is evaluated in the CFU assays.

Hematopoietic differentiation methods for PSCs can be used for disease modeling and therapy development for leukemias and other hematologic disorders, utilizing PSCs reprogrammed directly from patient cells (Imeri et al., 2023; Kotini et al., 2023). PSC-derived hematopoietic cells can also be cultured under lineage-specific conditions to induce further differentiation, yielding relatively pure populations of mature blood cells, including erythroid cells, megakaryocytes, monocytes/macrophages, and lymphocytes (Bernecker et al., 2019; Feng et al., 2014; Martins et al., 2023; Mesquitta et al., 2019; Shukla et al., 2017). Scaled-up versions of these lineage-specific cultures could potentially be used to produce large quantities of cells for transfusion and immunotherapy applications, provided that the yields and quality of these cells match those of primary hematopoietic cells. Ongoing research efforts are focused on enhancing the efficiency and fidelity of PSC-based hematopoietic differentiation protocols to achieve this goal.

Considerable research efforts have also been directed at method development to generate PSC-derived cells that can reconstitute hematopoiesis after transplantation. Most PSC-derived hematopoietic cells do either not engraft or only show transient myeloid-restricted engraftment after transplantation into immunodeficient mice. The formation of functional HSCs with long-term multilineage stem cell potential has until recently only been achieved with hematopoietic cells derived from genetically modified PSC lines that overexpress transcription factors regulating HSC self-renewal, such as HoxB4, RunX1, and SPi1, or via the formation of PSC-derived germ cell tumors, i.e. teratomas, in mice (Kyba et al., 2002; Sugimura et al., 2017; Suzuki et al., 2013). For practical and safety reasons, methods involving transgene overexpression or tumor formation are unsuitable for the development of clinically useful protocols. However, a recent study has demonstrated that HSCs with multilineage engraftment potential in immunodeficient mice can also be derived from PSCs without the need for transgene overexpression or teratoma formation. The culture conditions employed in this study promote the formation of complex structures akin to the hematopoietic environment supporting HSC formation in the human embryo (Piau et al., 2023). These promising findings underscore the capability of PSCs to differentiate into functional HSCs when exposed to appropriate microenvironmental signals.

Due to the complexity of the culture system, it may be difficult to fully harness the potential of PSCs as an effective cell source for HSC-based cell therapies. Further scientific advancements will be needed to enhance our understanding of the molecular mechanisms governing HSC development, refine and scale up PSC differentiation protocols, and realize the clinical potential of PSC-derived HSCs.

Summary And Conclusions

HSCs are multipotent cells that support the lifelong production of all blood and immune cells. They can be identified by their ability to reconstitute long-term hematopoiesis in transplantation assays, typically performed in mice. Hematopoietic progenitors, which are the intermediate stages between HSCs and blood cells, do not have long-term repopulating ability after transplantation and are measured in culture assays, such as the CFU assay, and LTC-IC assay. Flow cytometry and other antibody-based techniques further aid in the characterization and purification of hematopoietic cells by discerning expression patterns unique to HSCs, progenitors, and mature blood cells.

These assays are instrumental in advancing our comprehension of hematopoiesis and assessing the effects of ex vivo manipulations like purification, cryopreservation, culture, and drug exposure on HSPC numbers, viability, and function. Recent advancements in single-cell resolution techniques to measure chromatin structure, gene and protein expression, and metabolism have greatly enhanced HSPC characterization, offering profound insights into hematopoietic hierarchy dynamics and responses to stimuli.

While donor bone marrow, mobilized blood, and umbilical cord blood are the primary sources of HSPCs for research and clinical purposes, ex vivo expansion culture protocols have been devised to amplify the limited numbers of obtainable HSCs from these sources. The ability to expand HSPCs in culture is crucial for the success of clinical applications, including gene therapy, where sustained HSPC functionality in vitro is vital.

Furthermore, the advancement of technologies enabling the generation of HSPCs from pluripotent stem cells offers an alternative avenue for producing HSCs and blood cells for cellular therapies. Once technical hurdles are surmounted and functional HSCs and blood cells can be manufactured at scale, these innovative methodologies hold immense promise for the development of novel applications. These applications are anticipated to encompass the creation of universal donor cells suitable for transplantation or transfusion, thereby addressing limitations associated with donor availability and compatibility. Additionally, they may facilitate the engineering of immune cells tailored to combat challenging conditions, such as infections and cancers resistant to conventional treatments. By harnessing the potential of tissue-derived and pluripotent stem cell-derived HSPCs, future therapeutic strategies stand to revolutionize the treatment landscape and significantly improve patient outcomes.