A human body needs specialized cells to fight infection, to provide immunity against pathogens and to transport oxygen to tissues and organs. They include red blood cells, platelets and various types of white (immune) blood cells. Hematopoiesis is the process of producing, sustaining and replacing blood and immune cells that are destined to be destroyed by either programmed cell death or necrosis. The establishment and maintenance of the blood system start with a small number of hematopoietic stem cells (HSCs) in the bone marrow in adult mammals. These HSCs are self-renewing and can differentiate into more than ten cell types through a hierarchical pathway with increasingly restricted lineage potential. Cells at the top of this hierarchy have the potential to differentiate into multiple lineages (e.g. both red and white blood cells) while cells at the bottom of the hierarchy only differentiate to one cell type [1].

The highly regulated hematopoietic system ensures proper compensation of lost blood and immune cell types by sustaining their total counts. Adult humans generate more than two million red blood cells every second [2] and produce and removes about 10¹¹ platelets per day [3] ; around 10⁹ neutrophils per kg body weight leave bone marrow daily [4].

Hematopoiesis research utilizes mouse models extensively [5], largely due to ease of obtaining mouse bone marrows, and the findings in mouse models are often extended to human hematopoiesis (e.g. [6] ). Both mouse and human hematopoiesis rely on the HSCs at the top of the hierarchy to generate the different blood and immune cell types through a highly specialized, step-wise hierarchy. These hierarchical pathways of differentiation are shared by some progenitors e.g. the differentiation of mast cells and basophils follows a similar differentiation pathway in mouse and human hematopoiesis [5, 6]. Also, both mouse and human hematopoiesis occurs within the bone marrow microenvironment. However, mouse and human hematopoietic stem and progenitor cells express unique set of cell surface markers which serve as the guideline for isolation.

This review focuses on mature blood cell types and their differentiation pathways from HSCs. In addition, we will discuss how to isolate HSCs and other intermediate progenitor cells, especially laboratory protocols to generate the blood cell types from stem cells isolated from mouse bone marrow. Finally, the protocols for detection and analysis of these blood cell types will be highlighted.

Three main methods are currently employed to define and detect the differentiation of hematopoietic stem cells [7, 8].

• • In vivo transplantation into irradiated animals
• • In vitro colony forming assays
• • Inferring differentiation trajectories based on gene expression analysis

Traditionally, HSCs are defined by their ability to reconstitute or replenish the entire blood system of an immunocompromised recipient in a process called transplantation. In this process, animals (mouse models) are exposed to lethal radiations which destroy their whole blood system, making the animals vulnerable to infections. These animals (called recipient animals) then receives the bone marrow (including HSCs) from another immune-compatible animal (called donor animal). The HSCs of the donor animal will highjack the immune system of the recipient animal and begin producing the different blood and immune cell types [8]. Based on the different types of blood cells generated, the time duration and the ability to reconstitute the blood system in tertiary recipients, the different types of HSCs and their differentiation pathways are analyzed.

In addition to traditional in vivo transplantation methods, HSCs are also isolated from animals (via bone marrow) and humans (from cord blood samples) and put in liquid or semi-solid culture media with appropriate growth factors. After 8 - 10 days, the resulting colonies are analyzed and the different blood cell types are detected using cellular morphology, gene expression analysis, antibody staining or histological techniques [5, 9]. Here, based on the colony size and the types of mature blood cells generated, the different types of HSCs and their differentiation pathways are analyzed.

Very recently, global gene expression analysis at the single cell level (single cell RNA sequencing) was used to infer the differentiation pathways of HSCs. Here, all the cells from the bone marrow are isolated (with little prior knowledge) and subjected to single cell RNA sequencing. Computational algorithms are employed to detect the difference in gene expression in immature and mature cell types. This information is finally used to computationally infer the differentiation pathways of HSCs [7, 10].

[enlarge]

Figure 1. Differentiation pathways of hematopoietic stem cells.

The following are different types of murine HSCs and the intermediate progenitors as well as mature cell types and their functions. These cell types are still considered the standard in the field and their detailed overview can be found in the following articles [11, 12], see also Figure 1.

Hematopoietic stem cells (HSCs) reside at the top of the hierarchy and have the potential to generate all blood and immune cell types. These cells also have the ability to self-renew. These cells are further sub-divided into long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs). AE Rodriguez-Fraticelli et al identified transcription factor TCF15 as the key regulator of LT-HSCs [13]. Quite often, researchers may opt to use a mixture of stem and progenitor cells. For example, J Zeng et al enriched CD34⁺ hematopoietic stem and progenitor cells through a Miltenyi CD34 Microbead Kit to evaluate base editing of BCL11A erythroid enhancer [14]. Miltenyi CD34 Microbead Kit is a popular choice [15].

Multipotent progenitors (MPPs) are immediate downstream precursor cells of HSCs. Like HSCs, these cells have the potential to generate all blood and immune cell types. However, they lose the ability to self-renew.

Bipotent progenitors (e.g. GMPs or MEPs) are downstream precursors of MPPs but they lose the potential to generate all blood and immune cell types. Instead, these cells can only generate two different mature cell types e.g. megakaryocyte-erythrocyte progenitors (MEPs) which can only generate platelets and red blood cells but not macrophages etc. Like MPPs, these cells do not possess the ability to self-renew.

Unipotent progenitors (e.g. MkPs) are downstream precursors of bipotent progenitors but they lose the potential to generate two different cell types. Instead, these cell types can generate only one kind of mature cell type e.g. megakaryocyte-progenitors (MkPs) which can only generate platelets but not red blood cells. Like MPPs and bipotent progenitors, these cells do not possess the ability to self-renew.

Mature cell types lie at the bottom of the hematopoietic hierarchy and are functionally specialized cell types that can carry out diverse functions from transporting oxygen to the whole body to fighting infections. In addition to the main categories discussed below, there is significant heterogeneity among these cell types and are further sub-divided into different sub-types e.g. myeloid biased HSCs (My-HSCs) and lymphoid biased HSCs (Ly-HSCs) [16]. Mature cells can often be removed from analysis through direct lineage depletion kit from Miltenyi Biotec [17].

Red blood cells are generated in the bone marrow that contain hemoglobin and are responsible for the transporting oxygen to different tissues and organs of the body.

Megakaryocytes are giant cells with multiple nuclei and are produced in the bone marrow. These cells burst and give rise to thousands of platelets that reach the injury sites and aid in inflammatory reactions.

The progenitors of mast cells are generated in bone marrow and spleen, but mature mast cells are produced in specialized tissues e.g. intestines. These cells play a role in anti-allergic pathways.

Basophils are very close to mast cells in their origin and are generated in the bone marrow. These cells are responsible for the inflammatory reactions during immune response and results in the formation of allergic reactions.

Eosinophils are polymorphonuclear cell types generated in the bone marrow and circulate in the blood as well. These cells are associated with hypersensitivity and helminth infection.

Neutrophils are small white blood cells with multi-lobed nuclei. These cells ingest and destruct the microorganisms causing infections e.g. bacteria.

Macrophages are extremely large cells derived from monocytes. These cells secrete specialized chemical substances in response to foreign materials (e.g. bacteria) that activate the response of other immune cell types. These cells are also involved in phagocytosis of foreign material.

Dendritic cells process the antigen material (foreign material e.g. bacteria and protozoa) and present it to cell surface of T cells of immune systems.

B lymphocytes are lymphoid cells that produce antibodies. T lymphocytes are lymphoid cells that are responsible for cell- mediated immunity and trigger B cells to release antibodies in response to infections.

Hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow of mammals and express surface (membrane) proteins. Different HSPCs express a unique combination of these surface proteins (or surface markers) which allows for their prospective isolation from the bone marrow. Fluorochrome-conjugated antibodies against these surface markers are used to target specific HSPCs and with the use of fluorescence activated cell sorting (FACS) technique, different HSPCs are isolated (Table 1). In this section, the current standard of surface markers used to isolate different mouse HSPCs for research and diagnostic purposes are discussed [10]. Although huge literature is available with respect to the availability of surface markers of HSPCs, the sections below highlight the most widely used protocols in the field nowadays.

The most widely accepted protocols for isolating HSCs are reported [9, 18, 32]. A combination of CD34, CD48, CD135 and CD150 in addition to Sca-1 and c-Kit expression have been used. Long-term HSCs reside within lineage negative (see below), Sca-1^high and c-Kit^high fraction which accounts for < 0.1% of bone marrow. Also, HSCs are enriched within CD34^low, CD48^low, CD135^low fractions but highly express CD150 [9].

HSCs: Lineage^neg, Sca-1^high, cKit^high, CD34^low, CD48^low, CD135^low, CD150^high

Lineage cocktail includes markers of mature cells including B220 and CD19 (B cells), CD3e (T Cells), Gr-1 (myeloid cells), Ly-6G (neutrophils), Ter119 (erythroid cells).

In addition to the combination of above markers, these HSCs exhibit significant heterogeneity and are further sub-divided into 3 fractions using a combination of CD41 and CD150 as reported [16, 33]. Here, different HSC fractions are:

• HSCs (Fraction 1): Lineage^neg, Sca-1^high, cKit^high, CD34^low, CD41^low, CD150^high
• HSCs (Fraction II): Lineage^neg, Sca-1^high, cKit^high, CD34^low, CD41^high, CD150^high
• HSCs (Fraction III): Lineage^neg, Sca-1^high, cKit^high, CD34^low, CD41^low, CD150^low

Each of these fractions has a unique tendency to differentiate specifically into lymphoid and myeloid cell types [34].

In addition to these markers, EPCR has been reported to be marker highly enriched in long-term HSCs [35].

In the last 20 years, a huge number of studies reported the isolation of MPPs using different protocols and combination of surface markers. However, the protocol published [18] is the most widely adopted and the MPPs reported within are better characterized. This paper use a combination of CD34, CD48, CD135 and CD150 in addition to Sca-1 and c-Kit expression and sub-fractionates the classically defined MPP compartment into four different sub-populations with distinct differentiation bias towards megakaryocyte-erythrocyte, granulocyte-monocyte and lymphoid lineages [9].

• • MPP1 (also called short-term HSCs ): Lineage^neg, Sca-1^high, cKit^high, CD34^high, CD48^low, CD135^low, CD150^high
• • MPP2 (megakaryocyte-erythrocyte biased MPPs): Lineage^neg, Sca-1^high, cKit^high, CD34^high, CD48^high, CD135^low, CD150^high
• • MPP3 (granulocyte-monocyte biased MPPs): Lineageneg, Sca-1^high, cKit^high, CD34^high, CD48^high, CD135^low, CD150^low
• • MPP4 (lymphoid biased MPPs): Lineage^neg, Sca-1^high, cKit^high, CD34^high, CD48^high, CD135^high, CD150^low

The classical paper that first reported the isolation of granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythrocyte protenitors (MEPs) is Akashi et al [7, 9, 19]. The authors used a specific combination of CD16/32 and CD34 in addition to Sca-1 and c-Kit expression and resolved the progenitor compartment (Sca-1^low, cKit^high) into 3 fractions.

• • CMPs (common myeloid progenitors): Lineage^neg, Sca-1^low, cKit^high, CD34^high, CD16/32^low
• • GMPs (granulocyte-monocyte progenitors): Lineage^neg, Sca-1^low, cKit^high, CD34^high, CD16/32^high
• • MEPs (megakaryocyte-erythrocyte progenitors): Lineage^neg, Sca-1^low, cKit^high, CD34^low, CD16/32^low

However, the CMP compartment represents a huge mixture of myeloid cells and is currently not used anymore. Similarly, the MEP compartment represents a huge mixture of different megakaryocyte and erythrocyte progenitors also. Owing to the heterogeneity within different compartment, the protocols published in this report are rarely used these days.

To better resolve the CMP and MEP compartment, another protocol was reported in Pronk et al [9, 10, 20] which is currently the most widely used one. This paper describes a combination of CD16/32, CD41, CD150 and CD105 in addition to Sca-1 and c-Kit expression. The progenitor compartment (Sca-1^low, cKit^high) was resolved into distinct uni-potent and bi-potent megakaryocyte, erythrocyte and myeloid progenitors. The following populations are reported in this paper:

• • PreMegEs (bi-potent megakaryocyte-erythrocyte progenitors): Lineage^neg, Sca-1^low, cKit^high, CD41^low, CD16/32^low, CD105^low, CD150^high
• • MkPs (uni-potent megakaryocyte progenitors): Lineage^neg, Sca-1^low, cKit^high, CD41^high, CD150^high
• • PreCFUEs (uni-potent pre-colony forming unit erythrocyte): Lineage^neg, Sca-1^low, cKit^high, CD41^low, CD16/32^low, CD105^high, CD150^high
• • CFUEs (uni-potent colony forming unit erythrocyte): Lineage^neg, Sca-1^low, cKit^high, CD41^low, CD16/32^low, CD105^high, CD150^low
• • PreGMs (bi-potent pre granulocyte-monocyte progenitors): Lineage^neg, Sca-1^low, cKit^high, CD41^low, CD16/32^low, CD105^low, CD150^low
• • GMPs (bi-potent granulocyte-monocyte progenitors): Lineage^neg, Sca-1^low, cKit^high, CD41^low, CD16/32^high, CD150^low

Recently, with the aim of improving erythrocyte differentiation trajectory, Tusi BK et al resolved the progenitor compartment into distinct uni-potent and bi-potent megakaryocyte, erythrocyte and myeloid progenitors [10]. The following populations are reported in this paper:

• • P1 (uni-potent erythrocyte progenitors or CFU-Es) : Lineage^neg, Sca-1^low, cKit^high, CD55^high, CD49f^low, CD105^high, CD71^high, CD150^low
• • P2 (uni-potent erythrocyte progenitors or BFU-Es) : Lineage^neg, Sca-1^low, cKit^high, CD55^high, CD49f^low, CD105^high, CD71^low, CD150^high
• • P3 (basophil-mast cell progenitors): Lineage^neg, Sca-1^low, cKit^high, CD55^high, CD49f^high, CD105^low, CD41^low, CD150^low
• • P4 (uni-potent megakaryocyte progenitors): Lineage^neg, Sca-1^low, cKit^high, CD55^high, CD49f^high, CD105^low, CD41^high, CD150^high
• • P5 (bi-potent megakaryocyte-erythrocyte progenitors): Lineage^neg, Sca-1^low, cKit^high, CD55^high, CD49f^high, CD105^low, CD41^low, CD150^high

The primary source of erythropoiesis is fetal liver (during embryonic development) and bone marrow (in adults). One of the most widely used combination of surface markers to isolate progressively maturing erythroid cells includes CD71 and Ter119. This combination resolves the erythroid trajectory into 6 different populations [10, 21, 36, 37]. The following scheme shows the erythroid trajectory in the fetal liver (all fractions resides within the lineage negative fraction of fetal liver).

• S0 (immature progenitors): CD71^low, Ter119^low
• S1 (CFUEs): CD71^high, Ter119^low
• S2 (mixture of CFUEs and pro- erythroblasts): CD71^high, Ter119mid
• S3 (basophilic-erythroblasts): CD71^high, Ter119^high
• S4 (late basophilic and chromatophilic erythroblasts): CD71mid, Ter119^high
• S5 (orthochromatophilic erythroblasts and reticulocytes): CD71^low, Ter119^high

Similarly, another paper describes a combination of Ter119, CD44 and forward scatter profile (FSC) in FACS to resolve the erythroid trajectory [22].

• Pro-erythroblasts: CD44^high, Ter119mid
• Basophilic erythroblasts: Ter119mid, CD44^high, FSC^high
• Polychromatic erythroblasts: Ter119^high, CD44mid-^high, FSCmid
• Orthochromatic erythroblasts and reticulocytes: Ter119^high, CD44mid, FSC^low
• Red blood cells: Ter119^high, CD44^low, FSC^low

These protocols can be applied to both fetal liver and adult bone marrow erythroid differentiation trajectory. However, it should be kept in mind that bone marrow exhibits huge heterogeneity and impurities from other cell types compared to fetal liver. AE Rodriguez-Fraticelli et al defined erythroblasts with Ly6G⁻ CD19⁻ Ter119⁺ FSC^hi; [13].

Although a wealth of literature is available to isolate uni- and bi-potent monocyte, neutrophil and dendritic cell progenitors, Hettinger et al reported one of the widely accepted protocol using uses a combination of CD115, CD135, Ly6C and CD11b in addition to c-Kit expression to isolate these progenitors [23, 38]. The following populations are reported in this paper:

• • cMoP (common monocyte progenitors): Lineage^neg, CD115^high, cKit^high, CD135^low, CD11b^low, Ly6C^high
• • MDP (monocyte- dendritic cell progenitors): Lineage^neg, CD115^high, cKit^high, CD135^high, CD11b^low, Ly6C^low
• • Monocytes 1: Lineage^neg, CD115^high, cKit^low, CD135^low, CD11b^high, Ly6C^low
• • Monocytes 2: Lineage^neg, CD115^high, cKit^low, CD135^low, CD11b^high, Ly6C^high

Although, the granulocyte-monocyte progenitors (GMPs) were discussed in previous section, these progenitors can be further sub-fractionated into 3 different fractions with increasing bias towards monocyte and neutrophil lineages respectively using the protocol published in Yanez et al 2015 [9, 24, 39, 40].

• • GMPs (bi-potent granulocyte-monocyte progenitors): Lineage^neg, Sca-1^low, cKit^high, CD34^high, CD16/32^high, CD115^low, Ly6C^low
• • MPs (uni-potent monocyte progenitors): Lineage^neg, Sca-1^low, cKit^high, CD34^high, CD16/32^high, CD115^high, Ly6C^low
• • GPs (uni-potent granulocyte progenitors): Lineage^neg, Sca-1^low, cKit^high, CD34^high, CD16/32^high, CD115^low, Ly6C^high

Although, the bipotent progenitor with potential to differentiate into basophils and mast cells remain controversial, Qi et al reported their successful isolation using FceR1 marker in the GMP compartment (discussed above) [25, 41].

• • Pre-BMP (pre-basophil mast cell progenitors): Lineage^neg, Sca-1^low, cKit^high, CD34^high, CD16/32^high, FceR1^high

In addition, the another paper reported the isolation of uni-potent basophil progenitors [26, 42].

• • BaPs (basophil progenitors): Lineage^neg, cKit^low, CD34^high, FceR1^high

Similarly, Chen et al reported the successful isolation of uni-potent mast cell progenitors [27, 43].

• • MCPs (mast cell progenitors): Lineage^neg, Sca-1^low, cKit^high, Ly6C^low, FceR1^low, CD27^low, Integrin B7^high, T1/ST2 B7^high

The origin of eosinophils and their prospective isolation remain somewhat controversial. Iwasaki H et al reported the successful isolation of uni-potent eosinophil progenitors using a combination of CD34 and IL-5R [28, 44]. This paper used intermediate levels of c-Kit, rather than high or low levels, to isolate eosinophil progenitors.

• • EoPs (uni-potent eosinophil progenitors): Lineage^neg, Sca-1^low, cKit^mid, CD34^high, IL-5R^high

The existence of a common lymphoid progenitor giving rise to T cells, B cells and natural killer cells remained controversial for a long period. However, Kondo et al reported a population in the bone marrow that has the potential to differentiate into T cells, B cells and Natural killer cells but are devoid of myeloid differentiation [29].

• • CLPs (common lymphoid progenitors): Lineage^neg, IL7R^high, Thy-1^low, Sca-1^low, c-Kit^low

In addition to the stem cell and progenitor populations, the mature cells can be isolated using specific surface markers (or their combination). T cell markers and B cell markers are discussed in a separate article.

• Red blood cells: Ter119^high
• Megakaryocytes: DAPI^high, CD41^high (DAPI can be replaced with any nuclear dye since megakaryocytes possess multi-nuclei)
• Mast cells: c-Kit^high, FceR1^high
• Basophils: c-Kit^low, FceR1^high, CD11b^high
• Eosinophils: IL5R^high, CD11b^high
• Neutrophils: CD11b^high, Ly6G^high
• Monocytes/macrophages: CD11b^high, CD115^high, F4/80^high
• B cells: B220^high, CD19^high
• T cells: CD4^high, CD8^high, CD3e^high

Different researchers may use variations of the above combinations of markers. Y Chi et al used CD45⁺ CD3⁺ for T-lymphocytes, CD45⁺ CD11b⁺ Ly6G⁺ for neutrophils, CD45⁺ Nk1.1⁺ for NK cells, CD45⁺ CD11b⁺ (CD14⁺) Ly6C^high for monocytes and CD45⁺ CD11b⁺ (CD14⁺) Ly6C⁺ F4/80⁺ for macrophages [45]. AE Rodriguez-Fraticelli et al used Ly6G⁺ CD19⁻ Ter119⁻ for granulocytes and Ly6C⁺ Ly6G⁻ CD19⁻ Ter119⁻ for monocytes [13].

In the bone marrow, HSC niche regulates maintenance of stem cells. In addition to HSCs, cellular components of HSC niche are represented by mesenchymal stem cells (MSC), endothelial cells, osteoblasts, megakaryocytes, macrophages, adipocytes and lymphoid cells. In particular, MSCs are characterized by positive (CD73, CD90, CD105) and negative (CD14, CD19, CD34, CD45, HLA-DR) surface markers and the capacity to differentiate into other cell types. The interactions between HSCs and MSCs have been shown to be regulated via NF-κB signalling. Furthermore, loss of interaction with HSPCs could be partially repaired by exposing the MSCs to calcium ionophore or calmodulin [46]. Also, activation of Bmi1/Sirt1 signaling pathway in MSCs simultaneously promotes bone-building and hematopoiesis-supportive activities of osteoblasts in HSC niche [47].

Recent studies have shown that maintenance of MSCs is regulated via factors like BAP1 [48] and Emilin-2 [49]. In addition, MSC-derived exosomes (HP-MSC-DE) have recently been found to activate the Notch pathway on the membrane of CD133+ HSCs and enhance their proliferation [50].

Quiescence maintenance in HSCs is regulated by transcription factors, including HLF, Pbx1, Evi-1, Nurr1, Nrf2, C/EBPα, PU.1, YY1, Gfi-1, Fhl2, and Tcf15 [51, 52]. In addition, GATA transcriptional factors have been reported to be crucial for the growth of HSCs [53]. Among these factors, Gata2 inhibits proliferation and increases quiescence in HSCs by repressing gene expression of CCND1, CDK4, and CDK6, while Gata3 is essential for HSC entry into the cell cycle. Also, Lou et al. have shown that all-trans retinoic acid (ATRA) and inflammation upregulated retinoic acid-inducible gene I (RIG-I) in MSCs and RIG-I inhibition recovered the ATRA-treated stromal niche function to enhance HSC engraftment and emergency myelopoiesis for innate immunity [54].

In addition to the described transcription regulators of HSC quiescence, it has been shown that COP9 signalosome subunit 5 (CSN5), F-box and WD-40 domain protein 7 (Fbxw7) and Huwe1 maintain HSC quiescence by regulating ubiquitin-linked degradation of proteins involved in cell cycle regulation [55, 56]. Also, HSC cellular niches were found to require Runx1 or Runx2 transcription factors expressed by CXC chemokine ligand 12 (CXCL12)-abundant reticular mesenchymal cells to prevent niche fibrotic conversion and maintain HSCs and hematopoiesis [57].

Hematopoietic stem cells, intermediate multipotent progenitors, bi- and uni-potent progenitors can all differentiate to mature cells under carefully defined culture conditions. Although a wealth of literature is available, the most common conditions are discussed below. The basal culture conditions are composed of IMDM or serum-free expansion media including pen/strep (antibiotics to prevent infection), L-glutamine, beta-mercaptoethanol and lineage-specific cytokine cocktails. Some culture conditions are unique to different mature cell lineages while others are permissive culture conditions that support the proliferation and differentiation of different lineage-biased progenitors [5, 9, 10, 39].

Culture the sorted cells in IMDM medium with 20% FCS supplemented with SCF (50 ng/ml), IL-3 (10 ng/ml), IL-6 (10 ng/ml) and EPO (2 U/ml). In addition, the media include 1% pen/strep, 1x L-glutamine and 50 uM beta-mercaptoethanol. The culture condition supports the proliferation and differentiation of upstream progenitors into Ter119^high red blood cells. SCF, IL-3 and IL-6 support the basal proliferation of progenitor cells while EPO is crucial to the differentiation of red blood cells. The duration of the culture depends upon the sorted population e.g. MEPs or PreMegEs take 3 - 4 days to differentiate into red blood cells while HSCs and MPPs take upto 7 – 10 days [10].

Additional lineage specific cytokines can be added to derive other mature cell types, e.g. TPO (50 ng/ml) for megakaryocytes, IL-9 (50 ng/ml) for basophils and mast cells and IL-5 (10 ng/ml) for eosinophils.

Culture the sorted cells in IMDM medium with 20% FCS supplemented with SCF (50 ng/ml), IL-3 (10 ng/ml), IL-6 (10 ng/ml) and TPO (50 ng/ml). In addition, the media includes 1% pen/strep, 1x L-glutamine and 50 uM beta-mercaptoethanol. The culture condition supports the proliferation and differentiation of upstream progenitors into CD41^high multinucleated megakaryocytes. SCF, IL-3 and IL-6 supports the basal proliferation of progenitor cells while TPO is crucial to the differentiation of megakaryocytes. The duration of the culture depends upon the sorted population e.g. MEPs, PreMegEs and MkPs take 3 - 4 days to differentiate into megakaryocytes while HSCs and MPPs take upto 7 – 10 days [10].

Additional lineage specific cytokines can be added to derive other mature cell types, e.g. EPO (2 U/ml) for red blood cells, IL-9 (50 ng/ml) for basophils and mast cells and IL-5 (10 ng/ml) for eosinophils.

To differentiate the sorted cells to mast cells and basophils, culture the cells in IMDM or SFEM with L-glutamine, 20% heat-inactivated FCS, 1% pen/strept, and 0.1 μM beta-mercaptoethanol, supplemented with 20 ng/ml mSCF, 20 ng/ml mIL-3 and 50 ng/ml mIL-9 at 37°C, 5% CO2. SCF and IL-3 support the basal proliferation of progenitor cells while IL-9 is crucial to the differentiation of mast cells and basophils. The duration of the culture depends upon the sorted population e.g. PreBMPs, BaPs and MCPs take 3 - 4 days to differentiate into mast cells and basophils while MPPs take upto 7 – 10 days [5, 10].

Additional lineage-specific cytokines can be added to derive other mature cell types, e.g. EPO (2 U/ml) for red blood cells, TPO (50 ng/ml) for megakaryocytes, IL-5 (10 ng/ml) for eosinophils and GM- CSF (50ng/ml) for granulocytes and monocytes.

To differentiate the sorted cells to eosinophils, culture the cells in IMDM or SFEM with L-glutamine, 20% heat-inactivated FCS, 1% pen/strept, and 0.1 μM beta-mercaptoethanol, supplemented with 20 ng/ml mSCF, 20 ng/ml mIL-3 and 50 ng/ml mIL-5 at 37°C, 5% CO2. SCF and IL-3 supports the basal proliferation of progenitor cells while IL-5 is crucial to the differentiation of eosinophils. EoPs take 4 – 7 days to differentiate into eosinophils [5].

Additional lineage-specific cytokines can be added to derive other mature cell types, e.g. EPO (2 U/ml) for red blood cells, TPO (50 ng/ml) for megakaryocytes, IL-9 (50 ng/ml) for basophils and mast cells and GM- CSF (50ng/ml) for granulocytes and monocytes.

To differentiate the sorted cells to neutrophils, culture the cells in IMDM or SFEM with L-glutamine, 20% heat-inactivated FCS, 1% pen/strept, and 0.1 μM beta-mercaptoethanol, supplemented with 20 ng/ml mSCF, 20 ng/ml mIL-3 and 50 ng/ml G-CSF at 37°C, 5% CO2. SCF and IL-3 supports the basal proliferation of progenitor cells while G-CSF is crucial to the differentiation of neutrophils. GMPs take 3 - 4 days to differentiate into neutrophils.

In addition to G-CSF, GM-CSF or M-CSF can also be added which supports the simultaneous proliferation and differentiation of monocytes and macrophages. In order to derive other mature cell types, additional lineage specific cytokines can be added e.g. EPO (2 U/ml) for red blood cells, TPO (50 ng/ml) for megakaryocytes, IL-9 (50 ng/ml) for basophils and mast cells, IL-5 (50ng/ml) for eosinophils [5].

To differentiate the sorted cells to monocytes, culture the cells in IMDM or SFEM with L-glutamine, 20% heat-inactivated FCS, 1% pen/strept, and 0.1 μM beta-mercaptoethanol, supplemented with 20 ng/ml mSCF, 20 ng/ml mIL-3 and 50 ng/ml GM-CSF or 50ng/ml M-CSF at 37°C, 5% CO2. SCF and IL-3 supports the basal proliferation of progenitor cells while GM-CSF or M-CSF is crucial to the differentiation of monocytes and macrophages. GMPs take 3 - 4 days to differentiate into monocytes and macrophages [5].

Although M-CSF supports differentiation of monocytes and macrophages, GM-CSF supports both monocytes and neutrophils and can be added. In addition, G-CSF can also be added to yield neutrophils in the same culture. In order to derive other mature cell types, additional lineage specific cytokines can be added e.g. IL-9 (50 ng/ml) for basophils and mast cells and IL-5 (50ng/ml) for eosinophils.

To examine lymphoid colony formation, culture the cells in IMDM-based methocult M3630 supplemented with 100 ng/ml mSCF and/or 20 ng/ml FL at 37°C, 5% CO2. Progenitors are also cultured on irradiated S-17 stromal cell layers in 96-well plates with RPMI 1640 medium containing 10% FCS, 100ng/ml SCF and 10ng/ml IL- 7 [29].

Although a multitude of methods exist to detect and identify the mature cell types, the commonly employed methods include morphological analysis e.g. using May-Grunwald Giemsa staining. Gene expression analysis can be performed to detect lineage-specific genes. Alternatively, conjugated antibodies against lineage-specific surface markers can be added to the cultures followed by FACS analysis [5]. Finally, live imaging can also be performed as an alternative by adding color conjugated antibodies against lineage-specific surface markers followed by fluorescence microscopy [9, 39].

In the recent years, a good number of research articles reported the isolation of different hematopoietic stem and progenitor cell population (HSPCs) and the surface marker combination of different HSPCs evolve over time. Therefore, a thorough check and update on the literature is necessary. In addition, although the carefully selected surface marker combination enriches a specific HSPC population, the hematopoietic system suffers from a great deal of heterogeneity and impurity. Finally, the culture conditions reported here represent the commonly used standards for HSPC culture. Minor deviations exist across different reports and the researchers should test and optimize the conditions carefully.