IL-3/SCF/FLT3L: The Core Driving System for Efficient In Vitro Expansion of HSPCs

I. What Are Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are the core adult stem cells responsible for lifelong hematopoiesis in humans. As primitive hematopoietic cells present in bone marrow, peripheral blood, and umbilical cord blood, they serve as the “ancestors” of all blood cells. HSCs possess key characteristics including self-renewal, multipotent differentiation, and homing. Clinically, they are primarily used to treat hematologic disorders such as leukemia, lymphoma, and aplastic anemia, and also find application in the treatment of certain genetic diseases.

Figure 1. Hematopoiesis ^[1]

II. Applications of Hematopoietic Stem Cells

Hematopoietic stem cells act as the “seeds” of the human blood system, capable of generating all types of blood cells. Their core application is transplantation to rebuild healthy hematopoietic and immune systems.

Core Clinical Applications

• Hematologic Malignancies: Used for the treatment of leukemia, lymphoma, multiple myeloma, etc., by transplanting cells to restore normal hematopoietic and immune function.
• Benign Hematologic Diseases: Treatment of aplastic anemia, thalassemia, hemophilia, etc., by replacing defective hematopoietic cells to reinstate hematopoietic capacity.
• Genetic Diseases: Addresses congenital immunodeficiency diseases and inherited metabolic disorders by replacing defective cells with healthy hematopoietic stem cells to alleviate symptoms.
• Autoimmune Diseases: Used to treat systemic lupus erythematosus, rheumatoid arthritis, etc., by resetting the immune system to reduce abnormal immune responses.

Other Expanded Applications

• Adjuvant Therapy for Solid Tumors: For certain solid tumors (e.g., neuroblastoma), hematopoietic stem cell transplantation after high-dose chemotherapy rapidly restores bone marrow function and enhances treatment tolerance.
• Medical Research: Serves as a critical model for stem cell research, supporting fundamental studies on hematopoietic mechanisms, cell differentiation regulation, and disease pathogenesis, while also underpinning the development of cell therapy technologies.

III. In Vitro Expansion of Hematopoietic Stem Cells

During hematopoietic stem cell transplantation (HSCT), insufficient mobilization or the use of umbilical cord blood as the stem cell source often leads to a shortage of hematopoietic stem cells. This deficiency can lead to delayed engraftment post-transplantation, significantly increasing the risk of infection and bleeding, posing a serious threat to patient safety. Therefore, developing techniques that enable efficient ex vivo expansion of hematopoietic stem cells while preserving their self-renewal and multipotent differentiation potential holds critical clinical significance.

Figure 2. Components of ex vivo HSC culture systems ^[2] 

Core Technical Pathway Classification

• Endogenous Modification Pathway: Directly regulating HSC intrinsic properties, including gene editing modifications (editing stemness-related genes), small molecule compound-targeted regulation (acting on pathways like Wnt/Hippo), metabolic reprogramming (modulating glucose metabolism/mitochondrial metabolism), and epigenetic regulation (altering chromatin states).
• Exogenous Stimulation Pathway: Inducing expansion through external environments or substances, including cytokine combination induction (IL-3/SCF/FLT3L), biomimetic microenvironment culture (3D scaffolds/gel-simulated bone marrow niche), and co-culture with stromal cells (genetically modified stromal cells secreting hematopoietic factors).

Key Technical Characteristics

• Mainstream approaches predominantly employ a synergistic “endogenous regulation + exogenous stimulation” strategy, balancing expansion efficiency with stem cell maintenance.
• Clinical translation favors safer, controllable technologies like cytokine/small molecule combinations, while gene editing methods remain in preclinical research.
• Umbilical cord blood HSC expansion is a research hotspot; technical optimization can overcome cell number limitations and broaden transplant applicability.

Clinical Application Value

• Resolves mobilization failure and insufficient donor cells in umbilical cord blood during HSCT, reducing the risk of delayed engraftment.
• Improves transplant success rates for patients with high-risk hematologic diseases while minimizing complications like infections and bleeding.
• Provides an abundant source of functional HSCs for personalized cell therapy and regenerative medicine research.

IV. Optimization Strategies for In Vitro Expansion of Hematopoietic Stem Cells

To overcome technical bottlenecks caused by insufficient HSC collection, we employ multiple approaches on hematopoietic stem/progenitor cells (HSPCs) isolated from in vitro culture experiments. These include adding hematopoietic cytokines, screening activating compounds, co-culturing with stromal cells, simulating the bone marrow microenvironment with materials, and applying gene editing regulation. The goal is to achieve efficient HSC expansion while simultaneously maintaining their self-renewal capacity in vitro.

Cytokine Combination Induction

• The most fundamental approach, mimicking the in vivo hematopoietic microenvironment by adding multiple hematopoietic growth factors.
• Commonly used factors include stem cell factor (SCF), interleukin-3 (IL-3), FLT3 ligand (FLT3L), and thrombopoietin (TPO), which promote HSC proliferation either individually or in combination.
• Advantages include simplicity and high safety.

Biomimetic Niche Culture

• Constructs an in vitro environment mimicking the bone marrow niche, providing three-dimensional support and signaling regulation for HSCs.
• Common formats include 3D nanostructures, collagen gels, and bioengineered hydrogels, some loaded with cytokines or stromal cells.
• Significantly enhances HSC expansion efficiency and stemness maintenance.

Small Molecule Regulation

• Targets HSC stemness-related signaling pathways using small molecules.
• Representative compounds include Andrographolide, UM171, and SR1, which inhibit HSC differentiation and promote self-renewal.
• Advantages include lower cost, scalability, and synergistic enhancement of expansion effects when combined with cytokines.

Genetic Engineering Modification

• Modifies HSCs via gene editing technologies to enhance expansion capacity or stemness maintenance.
• Examples include modifying mesenchymal stromal cells to continuously secrete hematopoietic factors.
• High expansion efficiency, but involves safety and ethical considerations regarding gene editing; currently mostly in preclinical research stages.

Induced Pluripotent Stem Cell Differentiation

• Utilizes induced pluripotent stem cells (iPSCs) or embryonic stem cells as raw materials to generate functional HSCs through directed differentiation.
• Addresses the shortage of natural HSC sources, theoretically enables unlimited expansion, and supports personalized therapy.
• High technical difficulty requires precise differentiation process control; some approaches have entered early clinical trials.

V. IL-3/SCF/FLT3L Trio Unlocks HSPCs Expansion Potential

The IL-3/SCF/FLT3L trio represents a classic and highly efficient cytokine regimen for in vitro HSPCs expansion. By synergistically activating signaling pathways and regulating cell cycle progression, it significantly enhances expansion efficiency while preserving hematopoietic stem cell pluripotency, providing critical technical support for addressing cell shortage in transplantation.

Core Functions of Individual Factors

• IL-3 (Interleukin-3): Activates multiple signaling pathways including JAK/STAT and ERK to promote proliferation of multipotent hematopoietic progenitor cells, particularly eosinophils, basophils, and megakaryocytes. It enhances HSC responsiveness to other cytokines, demonstrating significant efficacy in repairing damaged HSCs (e.g., radiation injury) and reducing apoptosis.
• SCF (Stem Cell Factor): Initiates signaling by binding to the c-Kit receptor on HSC surfaces, propelling HSCs from the quiescent phase (G0) into the cell cycle to initiate proliferation. It sustains HSPCs survival and self-renewal, synergizing with IL-3 and FLT3L to amplify CD34⁺ cells.
• FLT3L (Fms-like tyrosine kinase 3 ligand): Targets the FLT3 receptor on HSC surfaces. While not independently proliferative, it synergizes with SCF and IL-3. It specifically maintains the undifferentiated state of early hematopoietic cells, preventing premature differentiation during HSC expansion and preserving more functional progenitor cells.

Synergistic Expansion Mechanism

• The three factors complement each other through “initiating proliferation + maintaining stemness + inhibiting apoptosis,” establishing a complete signaling network. SCF initiates the cell cycle, IL-3 enhances proliferation and survival signals, while FLT3L locks in primitive cell characteristics. Their combination significantly increases the proportion of cells positive for stemness markers such as CD34⁺CD38⁻.
• For HSCs from radiation-damaged or poorly mobilized sources, this combination effectively activates repair pathways, re-engaging surviving cells into the proliferation cycle. Its expansion efficiency significantly outperforms single-factor or other combination regimens.

Application Value and Advantages

• As a classic and mature protocol, it offers simple operation and high safety, widely applied for in vitro expansion of HSCs from diverse sources like cord blood and peripheral blood. It effectively overcomes limitations in primitive cell numbers.
• When combined with small-molecule compounds (e.g., UM171, SR1), it further amplifies expansion effects, providing ample functional cells for HSCT, shortening postoperative neutrophil recovery time, and reducing infection risks.

V. Yeasen HiActi™ Cytokines

Recombinant proteins are widely applied in core fields including stem cell and organoid culture, recombinant protein drugs, CAR-T cell therapy, and antibody drugs. With the rapid development of the biopharmaceutical industry, the recombinant protein market is growing rapidly, and demand for high-end raw materials is increasing year by year. To precisely match the continuously upgrading application needs in scientific research and industrial scenarios, and to address key pain points such as low activity and insufficient batch stability, Yeasen Biotech has built an innovative recombinant protein expression and purification platform based on years of R&D, production experience, and technological accumulation, focusing on providing high-activity recombinant protein products. To address challenges in HSPCs in vitro culture expansion, reduce expansion time, and enhance efficiency, Yeasen Biotech leverages its proprietary expression and purification platform to develop a series of HiActi™ cytokines—including IL-3, SCF, Flt3L, and other HiActi™ cytokines for HSPCs in vitro expansion. These products undergo rigorous quality control and cellular function validation to ensure high activity, high purity, high stability, and low endotoxin levels, thereby supporting hematopoietic stem cell research.

Product Data

Bioactivity of human IL-3

Figure 3. The ED₅₀ as determined by a cell proliferation assay using human TF-1 cells is less than 0.1 ng/mL, corresponding to a specific activity of > 1.0 × 10⁷ IU/mg.

Bioactivity of human SCF

 Figure 4. The ED₅₀ as determined by a cell proliferation assay using human TF-1 cells is less than 2 ng/mL, corresponding to a specific activity of > 5.0 × 10⁵ IU/mg.

Bioactivity of human FLT3L

 Figure 5. Measured by its binding ability in an ELISA method. Immobilized Human Flt3-Ligand at 1 μg/mL (100 μL/well) can bind Flt3-Ligand mouse monoclonal antibody. The ED₅₀ is 5.2 to 6.2 ng/mL.

Ordering Information

Product name	Item No.	Specification
Recombinant Human IL-3 Protein	90104ES	10μg/50μg/100μg/500μg/1mg
Recombinant Mouse IL-3 Protein	90143ES	2μg/10μg/50μg/100μg/500μg
Recombinant Human SCF Protein	92251ES	2μg/10μg/50μg/100μg/500μg/1mg
Recombinant Mouse SCF Protein	92260ES	2μg/10μg/50μg/100μg/500μg
Recombinant Human Flt3-Ligand/FLT3L Protein, His Tag	92279ES	10μg/100μg/500μg
Recombinant Mouse Flt-3 Ligand/FLT3L Protein	92610ES	10μg/100μg/1mg

 1. Mirantes C, Passegué E, Pietras EM. Pro-inflammatory cytokines: Emerging players regulating HSC function in normal and diseased hematopoiesis. Experimental Cell Research 2014; 329(2):248-254.

2. Wilkinson AC, Igarashi KJ, Nakauchi H. Haematopoietic stem cell self-renewal in vivo and ex vivo. Nature Reviews Genetics 2020; 21(9):541-554.