Stem Cell Research & Therapy, 02 May 2026
A new study published in Stem Cell Research & Therapy in 2026 successfully established a culture system for generating megakaryocytes (MKs) from CD34⁺ hematopoietic stem and progenitor cells isolated from human umbilical cord blood (UCB). The study also provided the first comprehensive single-cell transcriptomic characterization of these UCB-derived MKs. The findings demonstrated that laboratory-generated MKs closely resemble their physiological counterparts in morphology, function, and platelet-producing capacity, highlighting their potential as an alternative platelet source for future transfusion medicine.
Research Background
Platelets are indispensable components of the hematopoietic system, playing critical roles in hemostasis, coagulation, and immune regulation. However, the current platelet supply relies almost entirely on blood donors and remains insufficient to meet increasing clinical demand. Consequently, the generation of platelets from stem cells in vitro has emerged as a promising strategy to supplement platelet availability in the future.
Umbilical cord blood represents a rich and readily accessible source of hematopoietic stem cells. It is safe to collect, can be cryopreserved long-term in cord blood banks, and possesses significant regenerative potential. Nevertheless, the efficiency of generating MKs and platelets from UCB has not been fully investigated, particularly at the single-cell transcriptomic level.
Establishment of an Umbilical Cord Blood-Derived Megakaryocyte Production System
The researchers isolated CD34⁺ cells from human umbilical cord blood and cultured them in a two-stage differentiation system over 14 days.
During the first seven days, cells were expanded in StemSpan SFEM supplemented with IL-3 (20 ng/mL), stem cell factor (SCF, 50 ng/mL), IL-6 (50 ng/mL), and thrombopoietin (TPO, 50 ng/mL). From Day 7 to Day 14, the cells were transferred to medium containing TPO alone (100 ng/mL) to promote terminal megakaryocytic differentiation.
After 14 days of culture, approximately 2 × 10⁷ MKs were generated from an initial population of 1 × 10⁶ CD34⁺ cells. The proportion of mature CD34⁻CD41a⁺CD42b⁺ megakaryocytes reached approximately 70.0 ± 1.7%.
UCB-Derived Megakaryocytes Exhibit Native Megakaryocyte Characteristics
DNA ploidy analysis demonstrated a high degree of megakaryocyte maturation:
- 44.45 ± 3.28% of cells were diploid (2N).
- 31.2 ± 1.3% were tetraploid (4N).
- 15.4 ± 2.5% were octoploid (8N).
- 8.95 ± 1.27% displayed ploidy levels greater than 8N.
Wright–Giemsa staining confirmed the presence of diploid and tetraploid MKs, as well as thrombopoietic MKs actively producing platelets.
Transmission electron microscopy (TEM) further revealed hallmark ultrastructural features of mature megakaryocytes, including:
- Demarcation Membrane System (DMS)
- Open Tubular System (OTS)
- Alpha granules
- Mitochondria
These structures are essential for proplatelet formation and functional platelet release.
Robust Upregulation of Megakaryocyte-Specific Genes During Differentiation
Throughout differentiation, key megakaryopoiesis-associated transcription factors were progressively upregulated.
By Day 14:
- GATA1 increased 2.7-fold.
- NF-E2 increased 3.4-fold.
- FLI1 increased 5.1-fold.
- FOG1 increased 2.7-fold.
Notably, markers of megakaryocyte maturation exhibited striking increases:
- PF4 increased 415.9-fold.
- CD41 increased 8.9-fold.
- CD61 increased 30.5-fold compared with Day 4.
These findings indicate efficient differentiation and maturation toward the megakaryocyte lineage.
In Vitro-Generated Platelets Are Functionally Active
Platelets derived from UCB-MKs (UCB-PLTs) were stimulated with thrombin or TRAP6 to assess their activation potential.
Following stimulation, surface expression of CD62P increased markedly, indicating that UCB-PLTs retained the ability to respond to physiological activation signals similarly to native human platelets.
In clot formation and clot retraction assays, UCB-PLTs promoted fibrin clot formation and retraction at levels comparable to those of adult peripheral blood platelets, demonstrating functional hemostatic activity.
UCB-Derived Megakaryocytes Produce Platelets In Vivo
To evaluate their functionality in vivo, Day-14 UCB-derived MKs were infused into immunodeficient NCG mice.
Human platelets were detected in mouse peripheral blood shortly after infusion:
- 0.24 ± 0.04% at 30 minutes
- 0.27 ± 0.05% at 1 hour
- 0.23 ± 0.02% at 6 hours
- Nearly undetectable at 24 hours
These results demonstrate that UCB-derived MKs rapidly release functional platelets into the circulation following transplantation.
The investigators estimated that the average lifespan of the generated platelets was approximately six hours in this experimental model.
Single-Cell Transcriptomic Mapping Reveals Megakaryocyte Heterogeneity
Single-cell RNA sequencing of 8,064 cells collected on Day 14 identified nine transcriptionally distinct megakaryocyte subpopulations, designated MK1 through MK9.
Among these:
- MK1–MK8 represented conventional thrombopoietic megakaryocyte populations.
- MK9 expressed immune-associated genes such as NKG7 and CEBPA.
Gene set enrichment analyses revealed that MK9 shared molecular signatures with previously described immune megakaryocyte populations found in embryonic yolk sac and fetal liver tissues. These findings confirm the existence of an immune-related megakaryocyte subset that may perform functions beyond platelet production.
Umbilical Cord Blood Outperforms Human Embryonic Stem Cells as a Source of Megakaryocytes
Comparative analysis with megakaryocytes derived from human embryonic stem cells (hESCs) revealed significant advantages for UCB-derived MKs.
The proportion of actively proliferating megakaryocytes (active-cycling MKs) was substantially higher in the UCB-derived population:
- UCB-derived MKs: 35%
- hESC-derived MKs: 7%
Megakaryocyte generation efficiency was also markedly superior:
- UCB-MKs: 91.81%
- hESC-MKs: 26.37%
These findings suggest that UCB contains a larger proportion of early-stage megakaryocytes that retain strong proliferative and differentiation capacities, making it an attractive source for large-scale MK manufacturing.
Scientific Significance and Future Applications
This study demonstrates that CD34⁺ cells from human umbilical cord blood can be efficiently differentiated into mature megakaryocytes possessing the morphological, molecular, and functional characteristics of native MKs. Furthermore, it represents one of the first comprehensive single-cell transcriptomic maps of UCB-derived megakaryocytes, identifying nine distinct cellular subpopulations and revealing the presence of an immune-associated MK subset.
Collectively, these findings provide a critical foundation for the development of scalable megakaryocyte and platelet manufacturing technologies based on umbilical cord blood. Such advances may ultimately support the production of alternative platelet supplies for treating thrombocytopenia, improving hematopoietic stem cell transplantation outcomes, and advancing regenerative medicine applications.
References
He, Y., Liang, Y., He, J., Shen, Y., Wu, Z., Pei, S., & Zhu, F. (2026). Generation and single-cell characterization of functional megakaryocytes derived from umbilical cord blood. Stem Cell Research & Therapy.
Source: Stem Cell Research & Therapy
Link: https://link.springer.com/article/10.1186/s13287-026-05047-9
