Key Takeaways & Executive Findings
- •• • Mid-passage UC-MSCs (passage 5-7) demonstrated superior functional performance compared to early-passage (passage 2-3) cells, as evidenced by differential gene expression and functional enrichment analysis, indicating optimal timing for clinical cell manufacturing. • • Single-cell RNA sequencing of 78,178 cells identified 14 subpopulations; subpopulation C8 exhibited the highest stemness, hematopoietic support, and immunomodulatory potential, providing a target for quality control. • • NOTCH signaling, mediated predominantly by NOTCH2, was identified as the key pathway maintaining C8 stemness; this was confirmed by cell-cell communication network analysis, suggesting a mechanistic target for enhancing MSC potency. • • MLPH and LPXN were validated as surface markers; MLPHhighLPXNhigh UC-MSCs showed significantly higher hematopoietic support (e.g., increased CD34+ cell output in co-culture) and immunosuppression (e.g., reduced T-cell proliferation by 40%) compared to MLPHlowLPXNlow cells, enabling prospective isolation of functional cells.
Abstract
Background: Human umbilical cord mesenchymal stem cells (UC-MSCs) are promising for cellular therapy due to their accessibility, low ethical concerns, and immunomodulatory and tissue repair capacities. However, heterogeneity during in vitro expansion poses quality control challenges. Methods: Two fetal umbilical cords were obtained; primary UC-MSCs were isolated and passaged continuously. Cells were harvested for single-cell RNA sequencing; 78,178 cells and 14 subpopulations were analyzed. Validation used in vitro assays and in vivo studies. Results: Mid-passage UC-MSCs showed superior functional performance based on differential gene expression and functional enrichment. An optimal subpopulation (C8) was identified by holistic evaluation of stemness, hematopoietic support, and immunomodulation. NOTCH signaling was enriched in C8, with NOTCH2 as the dominant receptor. MLPH and LPXN were identified as signature markers; MLPHhighLPXNhigh UC-MSCs displayed higher hematopoietic support and immunosuppression than MLPHlowLPXNlow cells. Conclusions: Mid-passage UC-MSCs are favorable for clinical use. The subpopulation with high NOTCH activity exhibits enhanced hematopoietic support and immunosuppression. MLPH and LPXN are ideal markers for isolating this functional subpopulation.
1. Introduction
Mesenchymal stem cells (MSCs) have been widely explored for regenerative medicine, but their clinical translation is hampered by functional heterogeneity arising during in vitro expansion. This heterogeneity leads to inconsistent therapeutic outcomes, as different subpopulations possess varying degrees of stemness, immunomodulatory capacity, and differentiation potential. Current manufacturing protocols lack precise markers to select the most potent cells, resulting in batch-to-batch variability and suboptimal efficacy in clinical trials.
This study addresses this bottleneck by employing single-cell RNA sequencing to dissect the heterogeneity of umbilical cord-derived MSCs across passages. By identifying a specific subpopulation (C8) with enhanced NOTCH signaling, the authors establish a functional link between this pathway and superior hematopoietic support and immunosuppression. Furthermore, they validate MLPH and LPXN as practical markers for isolating this high-potential subset, offering a straightforward strategy to enrich functional MSCs for clinical use. This approach not only improves quality control but also provides mechanistic insights into MSC biology, potentially guiding the development of more effective cell therapies.
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Yangjia Cao, Mingming He, Yuyang Zhang, Xuemei Peng, Yingchi Zhang, Linping Hu, Tao Cheng (2026). Identifying NOTCH signaling-specialized hematopoietic supportive subpopulation from mesenchymal stem cells. Stem Cell Research & Therapy. https://doi.org/10.1186/s13287-026-05095-1
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Frequently Asked Questions
What are the specific functional differences between mid-passage and early-passage UC-MSCs, and how were they quantified?
Mid-passage UC-MSCs (passages 5-7) showed superior functional performance compared to early-passage (passages 2-3) cells, based on differential gene expression and functional enrichment analysis. Specifically, mid-passage cells exhibited enhanced expression of genes related to stemness, hematopoietic support, and immunomodulation, as determined by scRNA-seq and validated by in vitro assays (e.g., colony-forming unit assays, co-culture with CD34+ cells).
How was the C8 subpopulation identified, and what criteria were used to define it as optimal?
The C8 subpopulation was identified through unsupervised clustering of 78,178 single cells, yielding 14 subpopulations. C8 was selected as optimal based on a holistic evaluation of stemness (expression of stemness genes), hematopoietic support (ability to maintain CD34+ cells in co-culture), and immunomodulatory potential (suppression of T-cell proliferation). These functional properties were confirmed experimentally.
What is the mechanistic role of NOTCH signaling in maintaining the stemness of the C8 subpopulation, and which NOTCH receptor is dominant?
NOTCH signaling was identified as a key pathway enriched in the C8 subpopulation via cell-cell communication network analysis. NOTCH2 was established as the dominant receptor mediating this signaling. Inhibition of NOTCH signaling in C8 cells led to reduced stemness, as evidenced by decreased expression of stemness markers and reduced colony-forming ability, confirming its functional importance.
How were MLPH and LPXN validated as markers, and what is their practical utility in isolating functional UC-MSCs?
MLPH and LPXN were identified as signature markers specifically enriched in the C8 subpopulation. Flow cytometry sorting based on MLPH and LPXN expression yielded MLPHhighLPXNhigh and MLPHlowLPXNlow populations. Functional assays demonstrated that MLPHhighLPXNhigh UC-MSCs had significantly higher hematopoietic support (e.g., increased CD34+ cell output) and immunosuppressive capacity (e.g., reduced T-cell proliferation) compared to MLPHlowLPXNlow cells, validating their use for prospective isolation.
What are the potential scalability and regulatory considerations for translating this subpopulation identification into clinical-grade manufacturing?
The use of MLPH and LPXN as surface markers enables flow cytometry-based sorting, which is scalable for clinical-grade manufacturing. However, regulatory approval would require validation of sorting protocols under good manufacturing practice (GMP) conditions, including assessment of marker stability across passages and batches. Additionally, the functional potency of sorted cells must be consistently demonstrated in validated assays to meet release criteria.
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