Hematopoietic stem cell discovery: unveiling the historical and future perspective of colony-forming units assay

Hematopoiesis: a mirror of blood cells development

Stem cells are a distinctive group of cells found at all stages of life, capable of self-renewal and differentiating into various cell types. They play a central role in developmental biology and are essential for the repair processes that occur after injury or disease in the specific differentiated tissue they originate from Kolios & Moodley (2013). Stem cells have unique characteristics that distinguish them from fully matured cells, enabling them to differentiate into various specialized cell types based on their potency, differentiation capability, and origin (Poliwoda et al., 2022). This differentiation process is influenced by the category of differentiation potency that a stem cell possesses. There are different categories of potency associated with stem cells namely totipotent, pluripotent, multipotent, oligopotent and unipotent (Fig. S1) (Kalra & Tomar, 2014). HSCs are regarded as multipotent stem cells that are starting to show a more restrictive differentiation capacity which is limited to cell types from a single germ layer. This is indicated by the undifferentiated phenotype of HSCs that are capable of differentiating into common myeloid and common lymphoid precursor cells, followed by further differentiation into downstream lineage-specific and matured blood cells.

During embryonic development, blood cells originate from the yolk sac and later from the fetal liver and spleen. This early hematopoiesis is crucial for supplying the developing embryo with oxygen and nutrients. It mirrors the embryonic stages of development and contributes to the formation of the vascular system. In adults, hematopoiesis primarily occurs in the BM, particularly in the cavities of certain bones. This ongoing process reflects the body’s need for a continuous supply of mature blood cells to maintain homeostasis (Chen et al., 2020). Hematopoiesis involves the organization of stem cells, progenitor cells and differentiated blood cells. This hierarchy mirrors the hierarchical stages of development in which undifferentiated cells gradually give rise to specialized cell types (Wei & Frenette, 2018).

The HSCs niche refers to a complex microenvironment where HSCs and multilineage HSPCs are located. They migrate from their site of origin to the BM, where they establish a niche for continued hematopoiesis (Pinho & Frenette, 2019). This migration process reflects the movement of cells during embryonic development as they populate different tissues and organs. The BM microenvironment or niche plays a crucial role in regulating hematopoiesis. Hematopoiesis exhibits plasticity, allowing the adaptation of blood cell production in response to changing physiological needs (Yamashita et al., 2020). In addition, hematopoiesis is highly regulated by a complex network of signaling molecules, with cytokines, hormones and growth factors playing crucial roles in influencing various stages of hematopoietic development (Zhang & Lodish, 2008). Cytokines are signaling proteins that mediate communication between cells and they act as key regulators of hematopoiesis by controlling the self-renewal, differentiation, and maturation of HSCs and lineage-committed hematopoietic progenitor cells into functionally mature blood cells in the niche area (Pietras, 2017). The influence of cytokines on hematopoiesis is multifaceted and context-dependent.

Hematopoiesis starts from the asymmetric division of HSCs, where they produce new stem cells through self-renewal and also differentiation to produce progenitor cells that are multipotent progenitors (MPP) (Seita & Weissman, 2010). The progenitor cells will further divide to produce two types of progenitor cells, which are the myeloid, CMP or lymphoid progenitors, CLP. The CMP will conduct cell differentiation to produce two types of progenitors, namely the myelomonocytic, megakaryocyte-erythrocyte progenitors (MEP) and the myelomonocytic, granulocyte-monocyte progenitors (GMP), which will form erythrocytes/platelets and granulocytes/macrophages, respectively. Meanwhile, the CLP functions in the production of Pro-T, Pro-B and Pro-NK type progenitors from which these three types of progenitors can form mature white blood cells such as T cells, NK cells and B cells, respectively (Seita & Weissman, 2010) (Fig. S2).

HSPCs are able to give rise to different types of blood cells through the CFUs assay. The most frequently utilized in vitro assays are also known as the colony-forming cell (CFC) assays for assessing the proliferative and differentiative capacity of HSPCs into differential hematopoietic lineages (Sarma, Takeda & Yaseen, 2010). Technically, CFUs assays involve plating a single cell suspension of HSPCs at a designated cell density in a semi solid media, such as those based on methylcellulose with an addition of appropriate cytokines (Ezeh et al., 2016). Under these conditions, individual progenitor cells or CFUs are supported for proliferation and differentiation, leading to the formation of distinct colonies. These colonies, originating from various types of progenitor cells, are identified and tallied according to the number and kinds of mature cells they comprise, using morphological criteria (Kronstein-Wiedemann & Tonn, 2019). The CFUs assay is primarily employed to identify multipotential and lineage-restricted progenitors within the erythroid, granulocytic and macrophage lineages. It can also detect megakaryocyte and B-lymphoid progenitors when specific culture conditions tailored to these progenitors are used. While purified HSPCs are capable of forming colonies under suitable culture conditions, most CFUs found in the BM, blood, and other tissues consist of progenitors with limited self-renewal capacity and a constrained ability to repopulate hematopoietic cells in vivo (Sawai et al., 2016). However, the CFUs assay can act as a valuable substitute for HSPCs in situations where LT transplantation assays are either too costly or not feasible.

In essence, the CFUs assay can detect two types of erythroid progenitor cells namely the colony-forming unit-erythroid (CFU-E) and the burst-forming unit-erythroid (BFU-E) (Dulmovits et al., 2017). For the myeloid lineage, CFU-M (macrophage) and CFU-GM (granulocyte/macrophage) are differentiated based on the types of cells that make up the colonies they produce. Meanwhile the CFUs assay that is available for lymphoid progenitors is only limited for the detection of pre-B lymphoid progenitor cells (Radtke et al., 2019). An inverted light microscope can be used to observe colonies corresponding to lineage-specific HSPCs derived from CFUs assays. The morphological characteristics for each progenitor are described in Table S2 (StemCell Technologies, 2017). Since it was introduced more than forty years ago, the CFU assay has established itself as the standard in vitro functional test for investigating hematopoietic progenitor cells (HPCs). The CFUs assay is extensively utilized to examine the stimulating and inhibiting impacts of growth factors and to assess the effects of various in vitro interventions on cells. This includes processes such as cell handling, cryopreservation and gene transduction on cellular products employed in hematopoietic cell transplantation (Bradley & Metcalf, 1966). While more primitive HSCs mediate LT engraftment following transplantation, the quantity of CFUs in a graft has been linked to the efficiency of neutrophil and platelet engraftment and overall survival post-transplantation (Page et al., 2011). Therefore, the CFUs assay is a valuable tool for predicting the quality of a graft and has been especially effective in helping to select a higher number of functionally viable progenitor cells before HSPCs transplantation.

The past, current and future direction of colony-forming units assay in hematopoietic stem and progenitor cells research

The increasing capability to cultivate mammalian cells in vitro, combined with established clonal assays, facilitated the establishment of protocols that led to the development of in vitro hematopoietic CFUs assays, initially for mice cells and subsequently for human cells. The history of the CFUs assay in the context of animal and human HSPCs reflects the evolution of techniques used to study the clonogenic potential and differentiation capacities of these crucial cell populations. While the concept of CFUs has been known for several decades, the methods have undergone refinement over time. Therefore, the history of the CFUs assay is marked by a series of milestones that have shaped its application in both animal and human HSPCs research. From the early identification of CFUs to the incorporation of advanced technologies, the CFUs assay continues to be a fundamental and evolving tool in the study of hematopoiesis as shown in Fig. 1. Research in this area of interest has been ongoing since the 1940s and remains a subject of active investigation in the current scientific world.

The groundwork for the CFUs assay was laid with early observations of HPCs. In the late 1940s, studies involving BM transplantation in animals were conducted, providing initial evidence for the existence of cells within the BM capable of generating blood cells. During the 1950s, researchers observed that mice exposed to whole-body irradiation showed improved survival rates when their spleens were externally protected (Jacobson et al., 1951). In these experiments, protection was provided to one of the legs or femurs, a portion of the liver, or the intestine. They noted that while this led to increased survival, it was less effective than when the spleen was shielded. This highlighted the critical role of the immune system and later identified key sites for the growth and sustenance of hematopoietic cells (Jacobson, 1954).

Later, another study demonstrated the viability of BM transplantations by successfully transplanting BM from a mouse donor into irradiated mice recipients. Subsequently, they observed the formation of small lumps in the spleen, which led to the early discovery of colonies containing cells, identified as HSCs (Till & McCulloch, 1961). Additionally, it was also demonstrated that the number of spleen colonies correlated with the number of transplanted BM cells, indicating the presence of a specific subset of hematopoietic cells with the ability to restore hematopoiesis. This was the first evidence that elucidated the migration ability of transplanted HSCs to migrate to a specific niche within the BM microenvironment in the recipient body. Migration is a biological process that is the ability of the donor HSCs to migrate, engraft and proliferate in the recipient’s body to restore defective hematopoiesis, with the BM being their final destination. Their repeated experiments aimed to examine the resilience of healthy BM cells to radiation and provided cytological evidence of interactions between the cells (McCulloch & Till, 1962; Wu et al., 1968). Through these continuous experiments, the researchers were able to demonstrate that stem cells derived from the spleen colonies in BM are capable of generating new colonies, confirming the regenerative capacity of a single cell through direct cytology (Till & McCulloch, 1961; McCulloch & Till, 1962; Becker, McCulloch & Till, 1963; Siminovitch, McCulloch & Till, 1963). Thus, it is a cornerstone in understanding cellular biology and has significant implications in various fields, particularly in regenerative medicine and stem cell research.

Moreover, in the 1970s and 1980s, the CFUs assay gained prominence with the development of techniques to culture and identify hematopoietic colonies. The focus was initially on murine models, and researchers refined methodologies to isolate and study HPCs. They identified different types of CFUs representing various stages of hematopoietic cell differentiation. These included CFU-GEMM (granulocyte, erythrocyte, monocyte, megakaryocyte), CFU-GM (granulocyte-macrophage), and others. This added complexity and specificity to the assay. CFU-GEMM is defined as the most primitive HPC with the unique potency to form multilineage colonies consisting of granulocytes, erythrocytes, monocytes, and macrophages in the presence of specific growth factors such as IL-3 (Monette & Sigounas, 1987). Meanwhile, erythroid-committed progenitors will form two distinct types of colonies namely the CFU-E and BFU-E. In the presence of erythropoietin (EPO), CFU-E divides rapidly and gives rise to single erythroblast colonies, whereas BFU-E is generally a slow-dividing cell that gives rise to larger colonies of erythroblasts (Kimura et al., 1984). The immature slow-dividing BFU-E differentiates into intermediate mature BFU-E and then to fast-dividing CFU-E cells (Wu et al., 1995). CFU-GM is the progenitor cell for GM and gives rise to single lineage-committed progenitors, CFU-G (granulocyte) and CFU-M (macrophage), under the influence of colony-stimulating factors (CSF) such as GM-CSF, G-CSF, and M-CSF (Metcalf & Burgess, 1982). The biological process of generation of megakaryocytes and platelets is known as megakaryocytopoiesis. CFUs of megakaryocytes (CFU-Mk or CFU-Meg) are unipotent in nature and in the presence of thrombopoietin (TPO) give rise to mature megakaryocytic cells (Broudy et al., 1996; Kimura et al., 1984).

As the understanding of hematopoiesis expanded, the CFUs assay was adapted for human HSPCs. This development saw the establishment of techniques for isolating and culturing human hematopoietic colonies, paving the way for clinical applications. From these experimental findings, the field of clinical hematology embraced the practice of HSC transplantation by using BM cells as the main source to obtain HSCs. The earliest BM transplants were allogeneic, intended to restore the hematopoietic system in humans exposed to radiation following a nuclear accident. However, over time, the preferred way to harvest HSPCs have shifted to emerging sources such as mobilized peripheral blood-derived HSPCs and cord blood cells (Mathe et al., 1959). The CFUs assay became integral to HSCs transplantation. In the 1990s, it was employed to assess the quality and clonogenic potential of donor cells, ensuring successful engraftment. A study conducted by Page et al. (2011), showed that CFUs dose is a strong independent predictor of engraftment after unrelated umbilical cord blood transplantation (UCBT) in human and should be used to assess potency when selecting CBUs for transplantation. Cord blood banking also emerged during this period, with the CFUs assay being used to evaluate the efficacy of stored cord blood units (Skific & Golemović, 2019).

Before discussing umbilical cord blood banking, it is essential to understand that human blood, BM, body tissues, skeletal muscles, and embryos are important stem cells sources (Zakrzewski et al., 2019). The composition of umbilical cord blood is known to be 40% monocytes and 40% lymphocytes and the remaining 20% are neutrophils and progenitor cells (Theunissen & Verfaillie, 2005). In vitro studies have shown that CD34⁺ cells from umbilical cord blood (UCB) of human sample proliferate more rapidly than those from BM. Additionally, when UCB is transplanted in vivo, it demonstrates superior regulatory capabilities compared to BM stem cells (Theunissen & Verfaillie, 2005). With the help of CFUs assay, a defined seeding density can predict better cord blood potency.

Advances in cell biology, microscopy and molecular techniques have continuously improved the CFUs assay. Automation, high-throughput screening and single-cell analysis technologies have been integrated into the assay, enhancing its precision, efficiency and versatility. Counting cells and colonies via automated system is an integral part of high-throughput screens and quantitative cellular assays (Choudhry, 2016). Recent advancements in microfluidic technology have the potential to greatly enhance stem cell research by mimicking physiological environments to support the growth of stem cells. However, the use of microfluidic technology in CFUs application remains underreported which indicates the need for future exploration (Zhang & Austin, 2012). A critical method for evaluating their effectiveness and forecasting successful engraftment involves measuring the number and quality of lineage-specific progenitor cells and multipotent stem cells within the transplant population. Previously, a direct relationship between the quantity of HSPCs and engraftment success in human has been reported (Page et al., 2011). Commonly utilized indicators to determine the potency and quality of the graft include the total count of viable nucleated cells, the presence of CD34⁺ antigen-expressing cells, and the ability of cells to form distinct colonies of mature blood cells in semi solid growth media (Patterson et al., 2014). The adoption of an automated method would enhance both the precision and speed, thereby improving standardization in conducting the CFUs assay and result analysis (Pamphilon et al., 2013).

Assessing the type of CFUs based on morphological scoring which is conducted manually under microscopic observation involves subjective judgments about the shape, size, and other visual characteristics of colonies, which can vary depending on the observer’s experience and expertise. This variability can lead to human error and inconsistencies in data reporting, impacting the reliability of CFUs assessments. Hence, finding a new approach that could overcome this obstacle is essential to enhance accuracy and reproducibility in CFUs assay analysis, paving the way forward for successful experimental and therapeutic outcomes. One of the emerging technology-driven approaches that can be employed in CFUs assay is Artificial Intelligence (AI). By using AI-based technology, algorithms can be trained to recognize and count CFUs colonies through image processing. This would improve accuracy, eliminate subjectivity, and significantly speed up the analysis process (Kusumoto & Yuasa, 2019). AI systems could be designed to identify irregularities or anomalies in colony formation patterns, thus could help researchers to quickly identify issues such as contamination or unexpected changes in cell behaviour. AI can assist in integrating data from various sources, such as genetic information, cell signaling pathways and environmental factors, providing a more comprehensive understanding of the factors influencing CFUs formation. According to Kusumoto & Yuasa (2019), the primary domains within AI encompass computer vision, natural language processing (NLP), machine learning (ML), autonomous vehicles and robotics. ML is a type of algorithm that enables a computer to identify and categorize patterns within large data sets autonomously, without needing explicit programming for each task (Kusumoto & Yuasa, 2019). One specific method within ML, known as the convolutional neural network (CNN), falls under the category of supervised deep learning. CNNs have notably enhanced the accuracy of image recognition studies, demonstrating significant improvements in various research outcomes (Zeng et al., 2016). Deep learning-based approaches to analyze CFUs represent a significant advancement in the field of cell biology, particularly in the context of HSPCs research.

It has been demonstrated that AI technologies utilizing CNNs provide an effective platform for studying iPSCs, particularly in the analysis of colonies. As a result, the application of CNNs is poised to expand in the near future to encompass other types of stem cell analysis. HSCs could also benefit from these advancements in CNNs. Alongside iPSCs, HSCs represent one of the most established and widely utilized sources of stem cells for both medical and research purposes. HSCs play a critical role in maintaining the hematopoietic system (Butko, Pouget & Traver, 2016). They serve as a valuable resource for stem cell-based therapies and various hematological research studies. These studies span a wide range of applications, including toxicological assessments, developmental biology investigations, and drug testing endeavors (Ng & Alexander, 2017). Therefore, it is imperative to thoroughly investigate and validate the properties of HSCs using functional assays specific to HSCs.

Meanwhile, time-lapse microscopy allows for the continuous monitoring of colony growth, providing insights into dynamic changes in morphology over time. The capability to gather data on the quantifiable characteristics of progenitors as they form colonies offers a foundation for conducting mechanistic studies (Scanlon et al., 2022). For instance, this method can uncover the connection between cell cycle speed and fate specification as indicated by existing research, and it can assess the extent to which upstream progenitors engage in symmetric versus asymmetric self-renewal (Berg, 2014). Last but not least, quantitative morphological analysis such as digital pathology tools and image analysis software could be leveraged for quantitative analysis of hematopoietic CFU’s morphology. This includes observation of parameters such as size, shape, and density of the colonies.

CFUs assay can offer relatively simple in vitro model and require less sophisticated equipment compared to in vivo models, making them accessible for many laboratories. However, CFUs assay has limitations and is unsuitable to completely replace in vivo models. While it is an excellent tool for preliminary testing and screening, in vivo models remain essential for several reasons. In vivo models can assess both ST and LT HSPCs functionality, including self-renewal, differentiation, and engraftment. Besides, preclinical validation of therapies or drugs requires data from in vivo models to ensure safety and efficacy before advancing to clinical trials. Their models provide a more accurate representation of how cells behave in the context of a living organism, which is crucial for translational stem cell research. Thus, CFUs assay can only offer alternative and complementary models to study HSPCs in addition to in vivo models.

Overview of colony-forming units assay from different hematopoietic stem cell sources

A fundamental step in stem cell research and various medical applications is HSCs isolation. HSCs are the precursor cells that give rise to all the different types of blood cells in the body, making them vital for various therapeutic approaches, including BM transplantation and regenerative medicine. The isolation process involves the separation of HSCs from other cell types in a sample, such as BM, peripheral blood or cord blood and must be carried out with precision and care to ensure the purity and functionality of the isolated HSCs, which are essential for successful clinical outcomes and scientific advancements in the field of stem cell research (Morgan et al., 2017). HSCs can be obtained from different sources, such as BM, peripheral blood, and umbilical cord blood in humans (Lee & Hong, 2020). In animal models, HSCs are typically isolated from specific tissues, depending on the species used. The choice of source often depends on the intended application and the availability of suitable samples.

Stem cells are primarily classified into three types including embryonic stem cells (ESCs), adult stem cells and iPSCs. Each type has unique characteristics and possible applications. Both iPSCs and the CFUs assay are important elements in stem cell research. For example, iPSCs have the ability to differentiate into various cell types, including hematopoietic cells. Researchers can induce iPSCs to undergo hematopoietic differentiation protocols to generate cells of the blood lineage, including HSPCs. Usage of adult stem cells and iPSCs have become increasingly significant in medical applications, particularly for the regeneration and repair of damaged tissues due to their better availability and less ethical concerns as compared to ESCs (Seita & Weissman, 2010). Prior to stem cells application in research and therapies, it is crucial to accurately determine and verify their potency and function. This process requires trained scientists or operators for precise analysis. To date, CFUs analysis is performed through a fundamental technique that requires the need for microscopic observation of the colonies. Despite its importance, these analyses are typically carried out using traditional techniques (Ramakrishna et al., 2020). For example, the functional characterization of HSCs and iPSCs involves the microscopic identification and classification of colony subtypes through CFUs assays. By observing these colonies under a microscope, scientists can classify them based on their morphological characteristics, which reflect the types of cells they can differentiate into. This conventional method, which must be performed by a trained operator, is not only labor-intensive and time-consuming but also susceptible to inaccuracies in data reporting.

Prior to the CFUs assay, various techniques have been employed in sample preparation. For instance, the sample containing HSCs is collected and processed to remove any debris, red blood cells, and contaminants. This preparation step is crucial to obtain a pure population of HSCs. Depending on the specific application, the isolated HSCs may be cultured and expanded in vitro to obtain a larger population of cells for transplantation or research purposes. In many cases, a density gradient centrifugation is used to separate the mononuclear cell fraction, that contains HSCs from other blood components (Pösel et al., 2012). This technique relies on the differences in density among blood cell types, allowing the HSC-containing fraction to be isolated. Immunoselection is another common method for HSC isolation. It involves the use of specific antibodies that target unique cell surface markers expressed on HSCs (Jaatinen & Laine, 2007). By binding to these markers, the HSCs can be selectively separated from other cells using techniques such as magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) (Bacon et al., 2020). While FACS offers several benefits, including faster analysis and the ability to assess multiple parameters simultaneously, its drawbacks include the need for specialized equipment that is costly and the requirement for cells to be modified to exhibit a fluorescent feature (Shen et al., 2021). On the other hand, MACS avoids these drawbacks as it does not require specialist equipment or fluorescent labels. Instead, MACS utilizes magnetic particles that are functionalized to bind to specific cell subsets in a mixture, thereby enabling separation (Schmitz et al., 1994). The isolated HSCs are typically subjected to rigorous quality control measures to ensure their purity, viability and functionality. This may involve testing their differentiation ability and genetic integrity.

An overview of CFUs for various adult HSPCs models derived from humans and rodent/mice is shown in Table 1A. In addition, Table 1B addresses the CFUs for fetal sources from humans and rodent/mice while, Table 1C shows CFUs from other sources such as zebrafish and iPSCs. In vitro CFUs assays are commonly utilized in numerous studies to assess the quantitative and qualitative characteristics of HSCs. For example, Li et al. (2016) used the CFUs assay on BM samples from 365 patients that were recently diagnosed with myelodysplastic syndrome (MDS). The findings indicated that abnormalities in the proliferation and differentiation of erythroid and myeloid precursor cells in vitro mirror the ineffective hematopoiesis characteristic of MDS. These results could be valuable in forecasting outcomes for individuals with higher-risk MDS. A notable study in this area by Shpall et al. (2002), investigated the feasibility of using ex vivo expanded umbilical cord blood for transplantation. They utilized CFU assays to evaluate the growth and differentiation ability of cord blood-derived HSCs following ex vivo expansion, demonstrating the clinical utility of expanded HSCs in enhancing engraftment and reducing the time to hematopoietic recovery in human transplant recipients (Shpall et al., 2002).

Several studies in the 1990s used CFUs assays to show that embryos of mice exposed to benzene during pregnancy exhibited changes in the number of progenitor and hematopoietic precursor cells, along with an increase in granulopoiesis (Corti & Snyder, 1996; Keller & Snyder, 1988). More recently, our research has identified a lineage-specific response to benzene toxicity among HSPCs by using the CFUs assays (Chow et al., 2015; Chow et al., 2018). This research indicates that exposure to 1,4-benzoquinone (1,4-BQ) selectively reduces the clonogenicity of myeloid progenitors compared to lymphoid progenitors, highlighting the importance of lineage-specific mechanisms in the impact of benzene toxicity on the HSPC niche. Moreover, several other benzene metabolites studies also used the CFUs assay to form HSPCs colonies (Yi et al., 2018; Mohd Idris et al., 2018; Abd Hamid et al., 2020; Dewi et al., 2021).

A previous study by N’jai et al. (2010) used CFUs assay to demonstrate the rapid suppression (within 6 h) of lymphoid (CFU-PreB) and myeloid (CFU-GM) progenitor cells in 7,12-dimethylbenzathracene (DMBA) treated mice. The changes were not seen in Cyp1b1 null mice, indicating that local metabolism of DMBA in the BM by Cyp1b1 is necessary to affect BM CFU-PreB and CFU-GM. Additionally, these findings suggest that myeloid lineage cells recover more rapidly than lymphoid lineage cells following exposure to DMBA (N’jai et al., 2010). Meanwhile, the function of lymphoid progenitor cell in Down syndrome was observed using the CFUs assay. Examination of hematopoietic progenitor populations revealed that Ts65Dn mice had a reduced number of functional HSCs and a notably lower percentage of BM lymphoid progenitors (Lorenzo et al., 2011).

A number of studies have used the CFUs assays to grow fetal HSPCs. A notable study in this context is Ivanovic et al. (2004), who investigated the characteristics of HSCs within the early human embryo, focusing on their expression profiles and functional capacities. The study utilized CFUs assays to evaluate the clonogenic potential of isolated HSCs from fetal umbilical cord blood, demonstrating their ability to give rise to various hematopoietic lineages. Previous studies have shown that transplacental exposure to benzene in animal models such as C57BL/6N mice, increases reactive oxygen species (ROS) level in the fetal liver and finally affecting the colony counts of erythroid and myeloid progenitors (Badham & Winn, 2010; Badham et al., 2010). Meanwhile, exposing pregnant mice to benzene from gestation day (GD) 6 to 15 leads to persistent changes in fetal tissue hematopoiesis that last up to six weeks post-birth, as evidenced by the disrupted development of myeloid and erythroid progenitor cells (Keller & Snyder, 1986). Earlier research involving fetal samples revealed that HSCs derived from the yolk sac are more vulnerable to the cytotoxic effects of hydroquinone (HQ) compared to HSCs derived from adult BM when culturing using semi solid methylcellulose agar (Zhu et al., 2013).

Other sources of stem cells that have gained interest in relation to the usage of the CFUs assays are from zebrafish and iPSCs. Researchers have created a clonal, ST in vitro assay for zebrafish erythroid and myeloid progenitor cells using methylcellulose culture methods. This assay supported the growth of zebrafish hematopoietic progenitor cells and allowed for the quantification of progenitor numbers in adults, enhancing the understanding of HSPCs in zebrafish (Traver et al., 2003). Additionally, a study by He et al. (2015) who performed CFU-C assays on fetal liver-derived HSCs from Nlrc3^−/− embryos of zebrafish, revealed compromised colony formation abilities. Meanwhile, research involving the use of the CFUs assay in iPSC-derived HSC is a critical area within regenerative medicine and stem cell biology. Researchers have developed a novel method for generating functional HSCs from mouse iPSCs. CFUs assays were utilized among other techniques to evaluate the hematopoietic capacity of the derived cells, demonstrating their ability to differentiate into various blood lineages and reconstitute the hematopoietic system in recipient mice (Suzuki et al., 2013).

As discussed above, there are various HSCs sources and experimental models that require application of the CFUs assay for the study of HSCs and hematopoiesis. However, further exploration is required to establish a standard and optimal protocol of CFUs assay for the respective models and sources. Ongoing research on refining the CFUs protocol is fundamental as the outcome can improve and expand its application for various HSCs sources and experimental needs. The following section of this review will delve deeper into the impact of CFUs in both stem cell research and how it affects medical applications.

The impact of colony-forming units assay in stem cell research and medical applications

The CFUs assay plays a crucial role in stem cell research as it is a valuable tool for assessing the functional properties of stem cells. The CFUs assay is a powerful tool in stem cell research for assessing the lineage potential of individual stem and progenitor cells (Thompson et al., 2023). The CFUs assay has been used to study HSCs since its development in the 1960s, providing insights into their self-renewal, clonal differentiation and multilineage potential (Weissman & Shizuru, 2014). It has also been adapted to acquire data from single purified HSC populations, further advancing our understanding of the hematopoietic hierarchy (Skific & Golemović, 2019). The CFUs assay is considered the gold standard for assessing the potency of stem cell products, as it correlates with engraftment success and overall survival in recipients of HSCs transplantation (Velier et al., 2019). In the following sub-section, the impact of the CFUs assay in HSPCs analysis in relation to stem cells research and therapeutic application will be further described.

Unlocking the roles of colony-forming units assay in hematopoietic stem and progenitor cells industries

Stem cells have gathered significant interest and applications in various industries due to their unique ability to differentiate into different cell types and their suitability for regenerative medicine. The hematopoietic CFUs assay offers applications not only in research and clinical settings but also in the industrial context. Pharmaceutical companies use the CFUs assay to screen possible drug candidates that may affect HSPCs function. This is particularly relevant for drugs targeting hematological disorders or those with likely side effects on blood cell production. Besides, the CFUs assay helps to assess the potential toxicity of new compounds or industrial chemicals on HSCs, providing crucial safety information. Figure S3 shows a list of countries in relation to stem cell industries development up until 2020 (World Population Review, 2024). The United States leads with the highest number of trials, followed by South Korea, Australia and other countries, showcasing their respective contributions to the stem cell industry.

Companies involved in stem cell banking, including cord blood banks, use the CFUs assay to evaluate the quality of stored HSC samples. It helps ensure the clonogenic potential and viability of cells before storage or transplantation (Pamphilon et al., 2013). Industries focused on cell therapy, regenerative medicine and HSC-based therapies use the CFUs assay as a tool for assessing the potency and quality of therapeutic cell products, underscoring the importance of clonogenic potential in ensuring therapeutic efficacy (Yüksel et al., 2010). Furthermore, a study by Fernández-Santos et al. (2022) explored the optimization of cell culture conditions for HSCs using the CFUs assay. This research focused on the effects of various growth factors on the clonogenic potential of these cells, contributing to the development of more effective culture strategies.

Last but not least, ethical considerations are always a priority in stem cell research. The use of adult HSPCs instead of ESCs alleviates most ethical concerns that might otherwise arise. Adult HSPCs are typically derived from ethically non-controversial sources, such as BM, UCB, or mobilized peripheral blood. These sources do not involve the destruction of embryos or the creation of embryos specifically for research purposes, unlike ESCs (Seita & Weissman, 2010). The use of adult HSPCs reflects a commitment to advancing science while respecting ethical boundaries. It demonstrates that cutting-edge research can progress without crossing contentious ethical lines, showcasing a balance between innovation and responsibility.