GMP-Grade Recombinant Human TPO — Core Raw Material for In Vitro HSC Expansion and iPSC-Megakaryocyte Induction

With the rapid development of the cell and gene therapy industry, clinical-grade cytokines serve as crucial raw materials for in vitro cell expansion and differentiation. Their batch-to-batch stability, safety, and complete documentation chain directly dictate the process reproducibility and regulatory filing progress of clinical projects.

Targeting applications such as hematopoietic stem cell (HSC) in vitro expansion, megakaryocytic induction and differentiation, and iPSC hematopoietic differentiation, we officially launch GMP-grade Recombinant Human Thrombopoietin (Human TPO), providing a compliant and auditable cytokine raw material solution for preclinical research and IND filing stages.

TPO (Thrombopoietin) is the core hematopoietic growth factor regulating megakaryocyte proliferation, maturation, and platelet production. It also plays a pivotal role in hematopoietic stem and progenitor cell (HSPC) in vitro culture systems, frequently used in synergy with SCF, Flt3L, and IL-series cytokines to achieve in vitro expansion and directed differentiation of hematopoietic stem cells. It is widely applied in hematopoietic stem cell research, iPSC-to-hematopoietic cell induction, in vitro platelet generation, and cell therapy product preparation. Ordinary research-grade TPO is only suitable for basic laboratory research and falls short of clinical project raw material release and regulatory filing requirements regarding endotoxins, host residues, batch consistency, and documentation—making it a bottleneck for translating research into clinical practice.

1. Biological Functions of TPO

TPO is primarily synthesized and secreted by the liver, acting as the core hematopoietic cytokine regulating the megakaryocyte-platelet axis. Its receptor is c-Mpl, expressed on hematopoietic stem and progenitor cells, megakaryocytic lineage cells at all developmental stages, and the surface of platelets.

1) Regulation of Megakaryocyte Development & Platelet Production (Core Function)

Acts on bone marrow megakaryocytic progenitors, promoting megakaryocyte proliferation, differentiation, and maturation, and driving cell enlargement and polyploidization.

Induces cytoplasmic shedding of mature megakaryocytes to produce circulating platelets, directly regulating in vivo platelet number homeostasis.

Maintains peripheral blood platelet baseline; during thrombocytopenia, TPO levels rise compensatorily to stimulate platelet recovery.

2) Effects on Hematopoietic Stem Cells / Hematopoietic Stem & Progenitor Cells (HSPCs)

Supports HSC quiescent maintenance, survival, and self-renewal, preserving stemness.

In in vitro cultures, synergizes with SCF and Flt-3L to significantly promote CD34⁺ hematopoietic stem and progenitor cell expansion.

Not restricted to the megakaryocytic lineage: participates in multilineage hematopoiesis regulation and aids myeloid progenitor survival.

3) Effects on Platelets

Binds to c-Mpl on the platelet surface to regulate platelet lifespan.

Moderately enhances platelet activation sensitivity and participates in hemostasis-related signaling pathways.

4) Other Biological Effects

Participates in bone marrow reconstitution following hematopoietic injury, mediating hematopoietic recovery after radio-chemotherapy-induced myelosuppression.

An essential key factor for inducing iPSC differentiation toward megakaryocytes/platelets in in vitro iPSC hematopoietic differentiation systems.

Expressed in a small number of non-hematopoietic tissues, participating in partial tissue repair regulation, though not its primary function.

2. TPO Signaling Pathway

Pathway Activation Overview:

TPO binds to cell membrane c-MPL → induces c-MPL homodimerization and conformational change → intracellular Box1/Box2 binds and activates pre-associated JAK2 (core) and secondarily TYK2 → JAK2 autophosphorylation and phosphorylation of tyrosine residues in the intracellular segment of c-MPL → formation of SH2 domain protein recruitment anchors, simultaneously initiating 3 major downstream pathways: JAK2-STAT5/STAT3, RAS-MAPK-ERK, and PI3K-AKT.

1) JAK2-STAT Pathway (Core Pathway):

JAK2 phosphorylates receptor tyrosines, recruiting STAT5 (dominant) and STAT3;

STAT undergoes tyrosine phosphorylation and forms dimers to translocate into the nucleus.

Target genes include Cyclin D1, Bcl-xL, c-Myc, and P27.

Function

STAT5: Megakaryocytic progenitor proliferation, anti-apoptosis, and megakaryocytic maturation; maintenance of HSC self-renewal.

STAT3: More involved in stress-, inflammation-, and liver regeneration-related responses; phosphorylation indicators to detect: p-JAK2, p-STAT5(Y694), p-STAT3(Y705).

2) RAS-MAPK-ERK Pathway (Differentiation)

Receptor phosphorylation recruits the Grb2-SOS complex, activating Ras-Raf-MEK-ERK1/2.

Function: Promotes megakaryocyte differentiation, maturation, and proplatelet formation; prolonged stimulation leans more toward a differentiated phenotype; upregulates c-Myc to promote cell cycle progression.

3) PI3K-AKT Pathway (Survival & Metabolism)

JAK2 phosphorylates the receptor and recruits PI3K, generating PIP3 and activating AKT.

Function: Inhibits apoptosis, supports cell survival, metabolic reprogramming, and progenitor survival protection.

4) Negative Feedback Regulation (Pathway Termination)

SOCS1/SOCS3 (Primary): STAT nuclear translocation upregulates SOCS transcription; SOCS binds to JAK2/receptor, inhibits kinase activity, and mediates ubiquitin-proteasome degradation.

SHP1 Phosphatase: Directly dephosphorylates JAK2 and receptors, rapidly inhibiting signaling.

LNK and PIAS Proteins: Inhibit STAT transcriptional activity.

Receptor Endocytosis & Degradation: TPO-MPL complexes are endocytosed, TPO is degraded, downregulating signaling; platelets consume circulating TPO via the MPL 'sponge mechanism' to maintain systemic homeostasis.

 Figure 1 TPO Signaling Pathway

3. Core Application Scenarios

1) Hematopoietic Stem Cell (HSC) In Vitro Expansion

TPO is an indispensable core factor in HSC self-renewal and expansion culture systems, frequently combined with SCF, Flt-3L, and IL-6 to maintain stemness and promote proliferation for stem cell expansion system development. GMP-grade raw materials meet the safety and compliance requirements of cell therapy raw materials for IND/NDA filings.

 Figure 2 Components of the ex vivo HSC culture system ^[1]

2) In Vitro Platelet Generation

For in vitro preparation of functional platelets from iPSC/cord blood-derived stem cells, TPO is the core cytokine used for in vitro production of platelet-like particles and blood substitute R&D.

3) Megakaryocytic Differentiation

In vitro induction of CD34⁺ hematopoietic stem cells and iPSC-HSCs toward megakaryocytes, where TPO binds to the c-Mpl receptor to drive megakaryocyte proliferation, maturation, and polyploidization.

4) Hematopoietic Organoid Construction

Construction of bone marrow organoids and hematopoietic microenvironment models to simulate the in vivo hematopoietic microenvironment and study hematopoiesis and thrombopoiesis mechanisms.

5) Drug R&D and Pharmacological/Pharmacodynamic Evaluation

TPO receptor agonist drug screening: Used as positive controls for screening small-molecule TPO receptor agonists and antibody drugs.

Pharmacological models of thrombocytopenia: Evaluating the regulatory effects of candidate drugs on megakaryocyte proliferation and platelet production; applied in early pharmacodynamic evaluations of drugs for Immune Thrombocytopenia (ITP) and chemotherapy-induced thrombocytopenia.

Neutralizing antibody detection: Detecting anti-TPO neutralizing antibodies as reference standards in antibody drug development.

6) In Vivo Animal Experimental Applications

Used in dosing experiments with animal models (thrombocytopenia models) to evaluate in vivo thrombopoietic efficacy, pharmacokinetics, and toxicology.

4. Yeasen GMP-Grade Recombinant Human TPO Protein

This product is recombinant human TPO optimized and expressed using a high-efficiency eukaryotic expression system, fully retaining the protein's native spatial conformation and biological activity. The product specifically binds to the c-Mpl receptor and efficiently activates downstream signaling pathways. Unlike ordinary research-grade products, this product is manufactured, purified, quality-tested, and packaged in full compliance with GMP (Good Manufacturing Practice) guidelines for pharmaceutical products. It is fully traceable, avoiding batch-to-batch fluctuations, contamination risks, and regulatory compliance gaps associated with ordinary protein raw materials. It is a compliant core raw material adapted for preclinical research, drug applications, and industrial manufacturing.

1) Features

High Purity: >95%, avoiding immune reactions caused by host cell protein (HCP) or DNA residues.

Batch-to-Batch Consistency: Ensures stability and reproducibility of cell culture.

Compliance: Meets regulatory requirements for cell therapy products, supporting IND filings.

Animal-Origin Free (AOF): Free of animal viruses, pathogenic substances, and foreign agent contamination, ensuring high safety.

2) Adherence to Regulatory Guidelines

Complying with Chinese Pharmacopoeia (2020 Edition) Volume III General Rules: Quality Control of Raw Materials and Excipients for Biological Products Production.

Complying with USP Chapter <92> Growth Factors and Cytokines Used in Cell Therapy Manufacturing.

Complying with Ph. Eur. General Chapter 5.2.12, Raw Materials of Biological Origin for the Production of Cell-based and Gene Therapy Medicinal Products.

3) Quality Management System

GMP proteins are manufactured, tested, and released under a quality management system certified to ISO 13485:2016, ensuring inter-batch consistency and material traceability.

Provided with an approved Certificate of Analysis (COA).

Animal-origin-free materials and production environment.

Pharmaceutical raw and auxiliary materials production base.

Supporting online/offline audits.

4) Product Safety Quality Control System

Host DNA Residue: < 20 ng/mg

Host Protein Residue: < 0.5 ng/μg

Sterility Test: Negative

Mycoplasma Test: Negative

Exogenous Virus Test: Negative

5) Manufactured in a GMP ultra-clean production facility

Manufactured under a GMP system, rigorous Quality Risk Management (QRM) is executed throughout the entire process—from seed cell bank (MCB/WCB) establishment, upstream culture, downstream purification to final filling—ensuring the safety, consistency, and traceability of every batch.

6) Product Data

SDS-PAGE: Purity >95% as determined by SDS-PAGE under reduced conditions.

• Bioactivity: ED50 is less than 10 ng/mL as determined by a cell proliferation assay using human M07e cells.

 

7) Ordering Infomation

References

[1] Adam C Wilkinson, Kyomi J Igarashi, Hiromitsu Nakauchi. Haematopoietic stem cell self-renewal in vivo and ex vivo. Nat Rev Genet. 2020 Sep;21(9):541-554.