Summary of protein factors involved in hematopoietic stem cell culture

Introduction

In recent years, stem cells have become increasingly widely used in clinical applications. However, the proportion and quantity of stemcells in the human body are extremely low, which cannot meet clinical needs. Therefore, in vitro expansion and cultivation of stem cells have become increasingly important. Due to ethical and technological reasons, the treatment of stem cells still faces many problems. The surface markers of hematopoietic stem/progenitor cells and various lineages of blood cells are relatively clear, and the phenotypic characteristics of cells can be quantitatively selected, separated, and their functions can be relatively free, without the need for complex downstream processes such as "biological scaffolds" for nerve, vascular, and surgical transplantation. Therefore, it is the best model for stem cell expansion and differentiation, and is also convenient for direct clinical use.

Hematopoietic stem cells have a high degree of self-renewal and multiple differentiation potentials. They can produce all mature blood cells, such as red blood cells, white blood cells, platelets, and lymphocytes, and can rebuild the entire hematopoietic system. In vitro expansion of hematopoietic stem cells requires maintaining their self-renewal ability while also preventing their differentiation, making it a highly challenging technology. In recent years, a large number of experiments have shown that the differentiation of hematopoietic stem cells depends on cytokines. We will briefly summarize the factors and their effects used in hematopoietic stem cell culture.

Stem cell factor（SCF）

Stem cell factor (SCF) is a factor that acts by anchoring and expressing the tyrosine receptor c-Kit on the surface of all HSCs. Defective c-Kit expression leads to a decrease in the number of HSC expansion. Currently, almost all cytokine combinations used in HSC culture experiments contain SCF. In addition, both SCF and FL3 belong to the tyrosine kinase receptor TKR family, which has a synergistic effect on expanding primitive hematopoietic cells. By binding to specific TKR, SCF transmits signals to the cells, initiating early division and expansion of progenitor cells, allowing cells to begin expanding and inhibiting apoptosis after completing the G0 phase .

Thrombopoietin（TPO）

TPO was initially believed to be a specific growth factor for megakaryocytes, belonging to the category of specific acting cytokines that can maintain the expansion, differentiation, maturation, division, and formation of functional platelets in megakaryocytes. It is the preferred factor for expanding megakaryocytes. In recent years, experiments have confirmed that TPO plays an important role in promoting HSC expansion in vitro studies, and when combined with other cytokines, it can increase the total number of colony forming units and the expansion fold of CD34+ stem cells. Especially in FL3 combinations, it can maintain long-term growth and expansion of cord blood CD34+ stem cells.

interleukin-3（IL-3）

Interleukin-3 (IL-3), also known as mast cell growth factor, is a pleiotropic cytokine mainly produced by activated T lymphocytes, which can stimulate the proliferation and differentiation of pluripotent HSCs and different lineage oriented progenitor cells. After IL-3 induced heterodimerization of stem cell surface receptors, they can bind to many signal transduction proteins, such as the Janus kinase signaling transducer and transcriptional activator (JAK/STAT) pathway, thereby stimulating a downregulated signal flow and participating in the regulation of stem cell expansion . IL-3 can also activate the extracellular signal regulated kinase (ERK) pathway and the c-jun aminotransferase (JNK) pathway, inducing the growth, expansion , and survival of stem cells.

Interleukin-6（IL-6）

IL-6 is a multidirectional cytokine that plays an important role in host defense by regulating immune and inflammatory responses. IL-6 is produced by T cells, monocytes, fibroblasts, endothelial cells, and keratinocytes, and has multiple biological functions. It can promote B cell differentiation and antibody production, synergistically IL-3 play a role in the development of megakaryocytes and platelet production, induce the expression of acute phase proteins in the liver, and regulate bone metabolism. IL-6 transmits signals through the IL-6 receptor system, which is composed of two chains: IL-6Rα and gp130. STAT3 is the decisive molecule in maintaining the undifferentiated state of embryonic stem cells, while IL-6 is the initial promoter of the JAK/STAT3 signaling pathway.

LFLT3 ligand (FL)

FLT3 ligand is a growth factor that regulates early hematopoietic cell expansion . FLT3 ligand binds to cells expressing tyrosine kinase receptor FLT3. The FLT3 ligand itself does not stimulate the expansion  of early hematopoietic cells, but rather synergistically induces growth and differentiation with other CSFs and interleukins. Unlike SCF, FLT3 ligand has no effect on mast cells. Multiple subtypes of FLT3 ligand have been identified. The main bioactive form is anchored on the cell surface as the extracellular domain of transmembrane protein (209a.). The membrane bound isomer can be cleaved by proteins to generate biologically active soluble isomers.

FMS-liketyrosine kinease 3

The FMS-liketyrosine kinease 3 (FL3) highly expressed in CD34+CD38dim cells transmits signals to the cell by binding to its specific tyrosine kinase active receptors (TKRs). FL3 acts on HSC/HPC and exerts hematopoietic regulation by binding to TKR on the cell surface. FL3 is also a very important early progenitor cell stimulating factor, which has a significant promoting effect on the in vitro expansion of HSC/HPC. It can prevent CD34+stem cells from gradually differentiating and depleting HPC during in vitro expansion.

Transforming growth factor- β

Transforming growth factor- β、Transforming growth factor- β 1 β 2 and β 3 subtypes of mammals emit signals through the same receptor, causing similar biological responses. They are multifunctional cytokines that regulate cell expansion, growth, differentiation, and movement, as well as the synthesis and deposition of extracellular matrix. Transforming growth factor- β (TGF- β) is produced by bone marrow stromal cells.It inhibits early HSC/HPC from entering the S phase, causing most HSC/HPC to be in the G0 phase.

Macrophage inflammatory protein-1 α

Macrophage inflammatory protein-1β (MIP-1β) is a natural antagonist of MIP-1α, which be accompanied by it. It can relieve the inhibitory effect of MIP-1α on early HSC/HPC and prevent HSC from returning to a quiescent state.

p38

P38, as a signaling molecule belonging to the mitogen activated protein kinase (MAPK) family, inhibits the in vitro expansion of HSCs under normoxic conditions. Experiments have shown that when HSCs are added to serum-free culture media containing TPO, SCF, and FL3, oxidative stress activates p38 and p16, leading to a significant decrease in the number of mouse HSCs.

Granulomacrophage colony-stimulating factor (GM-CSF)

Granulocyte macrophage colony-stimulating factor is a drug used clinically for various causes of leukopenia or granulocytopenia. The current cell mobilization agent is granulocyte macrophage colony-stimulating factor (GM-CSF), which not only increases the number of hematopoietic stem cells in peripheral blood, but also assists in cardiac function and other functions.

Granulocyte colony stimulating factor (G-CSF)

The effects of granulocyte colony-stimulating factors generally include antigen presentation, enhancement of macrophage function, and promotion of hematopoietic stem cell expansion. Granulocyte colony-stimulating factor is a powerful mobilization agent for bone marrow stem cells, which can stimulate the expansion of autologous bone marrow stem cells and mobilize them from the bone marrow to peripheral blood.

Erythropoietin (EPO)

Erythropoietin (EPO) is the main stimulatory factor in hematopoietic differentiation, which can promote the differentiation of hematopoietic stem cells into primitive red blood cells, accelerate the division and expansion of young red blood cells, promote the synthesis of hemoglobin, and also play an important role in the study of red blood cell induced differentiation.

Hematopoietic stem cells have the potential for self-renewal and multi-directional differentiation. The combination of different factors has different effects on the expansion of hematopoietic stem cells. The use of hematopoietic stem cells can selectively induce the production of multiple cells, which undoubtedly provides new ideas for the expansion of NK cells. At present, NK cells can be induced from embryonic stem cells and iPSCs, but both embryonic stem cells and induced pluripotent stem cells need to be transformed into hematopoietic stem cells before they can differentiate into NK cells. Therefore, hematopoietic stem cells play an indispensable bridge in this process. The advantages of NK cells derived from stem cells are that they can be used on demand, have strong homogeneity, low cytokine release, and strong killing activity. Therefore, there is still a high enthusiasm for exploring NK cells derived from stem cells on the market. There are literatures on the induction of stem cells into NK cells. We mainly focus on the factors mentioned in the literature.

一、NK cells derived from human embryonic stem cells

hESCs were transferred to coculture with murine bone marrow stromal cell line M210-B4 in medium containing RPMI 1640, 15% defined fetal bovine serum , 2 mM L-glutamine, 1% nonessential amino acids , 1% penicillin/streptomyocin, and 0.1 mM  -mercaptoethanol with medium changes every 2 to 3 days as previously described. After 17 to 20 days, single-cell suspension was prepared and CD34+CD45+ cells were isolated, as previously described. Isolated cells were transferred to a second coculture with the murine fetal liver–derived stromal cell line AFT024 in medium containing a 1:2 mixture of Dulbecco modified Eagle medium/Ham F12, 20% heat-inactivated human serum AB, 2 mM L-glutamine, 1% penicillin/streptomyocin, 5 ng/mL sodium selenite, 50μM ethanolamine, 25μM -mercaptoethanol, 20 mg/mL ascorbic acid, interleukin-3, stem cell factor, IL-15 , Fms-like tyrosine kinase 3 ligand , and IL-7 . Cells were fed with fresh medium by half medium changes every 5 to 6 days. After 30 to 35 days in culture, cells were harvested, filtered through 70-μm filter, and used for further analysis. 

二、NK cells derived from human pluripotent stem cells

We harveste 18–21 days cells for CD34+ CD45+ progenitor cell enrichment .One hundred thousand CD34+ CD45+ cells were placed onto EL08-1D2 stroma with 1 ml of NK cell initiating cytokines (IL-3, IL7, IL-15, stem cell factor, and fms-like tyrosine kinase receptor-3 ligand ). NK cell cultures we refreshed with 0.5 ml of cytokine-containing medium every 4–5 days. Mature NK cells were measured at 28–35 days of culture on EL08-1D2.

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