Hematopoiesis from Pluripotent Stem Cells

Hematopoietic progression—survival, proliferation, or differentiation—is well-supported by cytokines and growth factors. The initial stages of pluripotent hematopoietic cell development is regulated by broadly acting cytokine groups/growth factors such as IL-3 (interleukin-3), SCF (stem cell factor), GMCSF (granulocyte-macrophage colony-stimulating factor), IL-1 (interleukin-1), IL-6 (interleukin-6), IL-11 (interleukin-11), IL-2 (interleukin-2), and thymosin beta-4. IL-3 and SCF support the survival and stimulate the proliferation of granulocyte progenitors. GMCSF acts as an autocrine mediator of cell growth. IL-1 plays an important role in hematopoiesis by stimulating marrow stromal cells to secrete CSFs (colony stimulating factors). Like IL-1, IL-6 also exhibits a wide variety of effects in hematopoiesis and the immune system as an acute phase protein, whereas thymosin beta-4 regulates cell life, differentiation, proliferation/growth, migration, and motility [1,5]. Like IL-6 and GCSF (granulocyte colony stimulating factor), IL-11 synergizes with IL-3 in stimulating the generation of colonies of human megakaryocytes in vitro. Therefore IL-11 may be an important regulator of megakaryocytopoiesis. In the hematopoietic hierarchical system, IL-2 along with SCF regulates promotes cytotoxic function by stimulating the proliferation and activity of NK (natural killer) cells (lymphoid stem cell→natural killer precursor→natural killer cell) [4].

Differentiation of HSCs to lineage-specific primitive progenitor cells like myeloid stem cells (IL-3 and GMCSF) or lymphoid stem cells (IL-3) permits proliferation of the committed progenitor cells into other categories of major marrow cells. The various progenitor cells are identified by the type of colony they form. In culture media, the progenitor cells are defined as CFUs (colony forming units). The earliest detectable hematopoietic progenitor cell that rise to granulocytes, erythroblasts, monocytes, and megakaryocytes is termed CFU-GEMM (colony-forming unit granulocyte erythrocyte monocyte macrophage). Physiological regulation of myeloid stem cells into CFU-GEMM is mediated by IL-3, IL-6, IL-1, SCF, GMCSF, and IL-12 (interleukin-12). CFU-GEMM mature into more specialized precursor cells termed as CFU-GM or CFU-C (colony-forming unit–granulocyte and macrophage/monocyte or colony-forming unit in culture); CFU-Eo (colony-forming unit–eosinophil); CFU-Bas (colony-forming unit–basophil); CFU-Mast or CFU-MC (colony-forming unit–mast cell); CFU-E (colony-forming unit–erythroid) and CFU-Meg or CFU-Mk (colony-forming unit–megakaryocyte). The burst-forming units BFU-E (burst-forming unit-erythroid) and BFU-Meg or BFU-Mk (burst-forming unit–megakaryocyte) are earlier erythroid progenitors than the CFU-E and CFU-Meg, respectively. The term “burst” is significant of mitotic activity of CFU-stem cells to form a very large number of subsequent progenitor cells. Such proliferation of CFU-GEMM is promoted by IL-3, GMCSF , SCF, IL-5 (interleukin-5), IL-6,IL-11, IL-12, LIF [leukemia inhibitory factor (cholinergic differentiation factor)], and Epo (erythropoietin) [5,6]. IL-12 acts as a key modulator of immune function. It also acts as a growth factor for activated T and NK cells, enhances the lytic activity of NK/lymphokine-activated killer cells, and stimulates the production of Ifn-Gamma (interferon-gamma). Epo primarily regulates levels of erythrocyte progenitors and has pleiotropic effects both within the hematopoietic system and in other tissues [5]. LIF is responsible for cell life, proliferation, and growth, whereas GMCSF, IL-5, SCF, IL-3, and IL-6 enhance proliferation and differentiation of granulocyte progenitors into mature cells; specifically IL-5 in the case of eosinophils, SCF in the case of basophils or mast cells, and GCSF/GMCSF in the case of neutrophils [4].

Several of the CSFs stimulate colony formation of progenitors in vitro and have a broad spectrum of overlapping activities. During erythroid developmental progression, BFU-E, IL-3, GMCSF, and Epo have profound stimulatory effects on precursors cells at various stages. Epo is mainly responsible for differentiation of proerythroblasts to erythrocytes. Likewise, the process of megakaryocytopoiesis (BFU-Meg, IL-3, and GMCSF) with Tpo (Thrombopoietin) acts as a regulator of the megakaryocytic lineage, while IL-6 stimulates the formation of platelets from megakaryocytes. It has a particular role in platelet production, but is not essential for this. CFU-Mast differentiates as mast cells after cell activation in response to SCF and IL-3, also to IL-4 (interleukin-4), IL-5, and IL-9 (interleukin-9). IL-4 enhances colony formation by primitive hematopoietic precursor cells while IL-9 serves as a regulator of both lymphoid and myeloid systems. It supports IL-2 independent and IL-4 independent growth of helper T-cells and plays a crucial role in hematopoiesis [6,7]. Basophils arise from CD34+ progenitor cells found in cord blood, peripheral blood, and bone marrow. Basophilic differentiation is determined by CFU-Bas IL-3, IL-4, and GMCSF. In particular, cells that are CD34+ and express the receptors for IL-3, IL-4, IL-5, and GMCSF are considered eosinophil-basophil progenitors. Eosinophil colony differentiation is determined by CFU-Eo IL-3, IL-5, GMCSF, and IL-4. IL-4 predominates the initial phase of this differentiation. Eosinophil colony formation is stimulated also by IL-7 (interleukin-7), synergizing with IL-3 and GMCSF; this supportive effect on precursors of eosinophils is mediated by the endogenous release of IL-5. Interleukin-7 through interleukin-15 play vital roles in hematopoiesis and immune development and function [5,7]. Further myeloid stem cell differentiation into granulocyte progenitors is promoted by IL-3, GMCSF, and GCSF,giving rise to CFU-G (colony-forming unit–granulocyte) and CFU-M (colony-forming unit–macrophage). GMCSF strongly synergizes with GCSF in the formation of colonies with the appearance of granulocytes and enhances the in vitro survival of CFU-G. IL-3 does not stimulate granulocytic colony formation by itself but appears to regulate the survival and proliferative rate of progenitors of granulocytes. GCSF stimulated growth of CFU-G is further stimulated by IL-4. CFU-G ultimately matures into polymorpho-nucleated neutrophils [CFU-G IL-3, IL-4, GMCSF, GCSF, LIF (colony-stimulating factor–macrophage-specific) and IL-8]. LIF regulates the monocytic and granulocytic lineages and IL-8 promotes cell proliferation/growth of monocytes to neutrophils [3,4,8].

Pluripotent hematopoietic stem cells thus differentiate into bone marrow as myeloid or lymphoid stem cells. Myeloid stem cells give rise to a second level of lineage-specific CFU cells that go on to produce neutrophils, monocytes, eosinophils, basophils, mast cells, megakaryocytes, and erythrocytes. Monocytes differentiate further into macrophages in peripheral tissue compartments. Lymphoid stem cells, on the other hand, give rise to B-cell, T-cell, and NK cell lineages [9]. IL-1, IL-2, IL-6, IL-7, and SCF act on multipotential lymphoid stem cells, which further differentiate into specific B-cell and T-cell progeny. Depending on TCR (T-Cell Receptor) gene arrangements, T-cell progenitors develop as T-cells with TCR-Gamma/TCR-Delta positive receptor and TCR-Alpha/TCR-Beta positive receptor. The TCR-Alpha/TCR-Beta positive TCPs (T-cell Progenitors) are CD4 , CD8, and TCR negative and under the action of IL-2, IL-4, IL-7, IL-9, and IL-10. IL-7 is crucial for T-cell development and IL-10 is a pleiotropic cytokine with important immunoregulatory functions whose actions influence activities of many of the cell-types in the immune system. The lineage-committed TCPs give rise to T-Helper (TH) cells (IL-2, IL-7 and IL-12) and Cytotoxic (T_c) T-cells (IL-2, IL-5, IL-7, and IL-12). The basic difference between cytotoxic and helper T-cells is that all the TH cells are CD4+ and all Tc cells are CD8+ [5,8,9]. The process of T-Helper cell differentiation further gives rise to T-Helper cell subsets like T_reg (or regulatory T-Cells which are CD4+, CD25+, and FoxP3+); TH3 (adaptive T_reg cells); TH1 (chiefly mediates cellular immune response); TH2 (chiefly mediates humoral immune response); TH17 (marked by IL-17 cytokine release), and THi (T-Helper Intermediate); whereas, antigen stimulation leads to development of memory T-cells [10].

Similarly, cytokines are also crucial for development of B-cell progeny. The lymphoid stem cells first develop into B-cell progenitors or BCPs (IL-1, IL-6, IL-7, and SCF); BCPs give rise to pro B-cells (IL-6, IL-7, IL-11, IL-12, LIF, GCSF , SCF, and IL-3) and pro B-cells develop into mature B-cells, with IgM (immunoglobulin-M) or IgD (immunoglobulin-D) as surface receptors. Cytokines like IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, Ifn-Gamma, TGF-Beta (transforming growth factor-beta) regulate isotype switch signals that differentiate mature B-cells into switched plasma cells secreting non-IgM, IgM antibody secreting B-cell, and memory B-cells (activated upon antigen stimulation). Here IL-4 is crucial for differentiation of BCPs into B-cells. IL-11 stimulates the T-cell dependent development of IgG (immunoglobulin-G)-secreting B-cells. IL-13 is critical in regulating inflammatory and immune responses involved in several stages of B-cell maturation and differentiation, down-regulating macrophage activity, and inhibiting the production of pro-inflammatory cytokines and chemokines. Ifn-Gamma is produced mainly by T-cells and NK cells activated by antigens, mitogens, or alloantigens. It is produced by lymphocytes expressing the surface antigens CD4 and CD8. B-cells also produce Ifn-Gamma. TGF-Beta, being a multifunctional peptide, controls proliferation and differentiation among other functions in many cell types. Plasma cells and B-cells secrete immunoglobulin molecules like IgM, IgD, IgG, IgA (immunoglobulin-A) and IgE (immunoglobulin-E), essential for humoral immune response [5,8].

Per day, hematopoiesis yields approximately 175 billion red cells, 70 billion granulocytes (neutrophils, eosinophils, basophils), and 175 billion platelets. Thus, every day, billions of new blood cells are produced in the body, each one derived from a single hematopoietic stem cell. Colony-stimulating factors, cytokines, and cytokine receptors provide signals that enable the survival and proliferation of the multipotential and mature hematopoietic cells [5]. The immune system uses many mechanisms to combat infection by microbes. The fully integrated immune response draws elements from many effector systems to tailor a response to the specific invading pathogen. Hence abnormal regulation of the various effector mechanisms may lead to chronic or acute tissue damage. Understanding the relationships between different immune effector pathways may permit improved immunomodulatory therapeutics, development of improved vaccines, and avoidance of unintended tissue injury [9]. Cytokines may act as oral agents to stimulate hematopoiesis and become part of routine care in subjects with anemia, neutropenia, thrombocytopenia, or all these conditions as a result of natural or iatrogenic causes [4].