RUNX1 is a Key Inducer of Human Hematopoiesis Controlling Non-Hematopoietic Mesodermal Development

The Dominant Role of RUNX1 in Human Hematopoietic Development and Mesodermal Fate Balance — An Interpretation of “runx1 is a key inducer of human hematopoiesis controlling non-hematopoietic mesodermal development”

1. Academic Background and Research Motivation

The development of the hematopoietic system is fundamental for the growth and maintenance of higher organisms. Previous studies in mouse models have shown that the transcription factor RUNX1 (Runt-related transcription factor 1, also known as AML1/CBFA2) is indispensable in the process of “definitive hematopoiesis”. While there are some mechanistic similarities between human and murine hematopoiesis, notable differences exist—particularly in early hematopoiesis, regulatory pathways at each differentiation stage, and the fate determination of pluripotent stem cells. Until now, many mysteries remain unsolved regarding the precise role of RUNX1 in early human hematopoiesis and the mechanisms by which it regulates non-hematopoietic mesodermal fates.

In addition, gene mutations or rearrangements at the RUNX1 locus are frequently detected in malignant hematologic diseases, such as acute myeloid leukemia (AML). The deeper mechanistic role of RUNX1 in the fate decision of hematopoietic cells and the pathogenesis and progression of malignant blood disorders urgently needs clarification. Importantly, there has been virtually no report concerning the regulatory role of RUNX1 in the development of non-hematopoietic mesodermal cells (such as mesenchymal stem cells (MSCs), vascular endothelial cells, etc.) in humans.

Against this background, the authors of this study conducted a systematic investigation—aiming, through genetic manipulation and molecular biology approaches, to uncover the critical role of RUNX1 in guiding early human hematopoietic development and suppressing non-hematopoietic mesodermal fate, as well as dissecting the upstream and downstream regulatory pathways and branching molecular networks involved in the balance of developmental fate.

2. Paper Source and Author Information

The research paper is titled “runx1 is a key inducer of human hematopoiesis controlling non-hematopoietic mesodermal development,” published in 2025 in Stem Cells (Oxford University Press, Volume 43, Issue 5, Article sxaf019, DOI: 10.1093/stmcls/sxaf019). The leading authors include Zahir Shah, Cuihua Wang, Hanif Ullah, Hao You, Elena S. Philonenko, Olga V. Regan, Pavel Volchkov, Yong Dai, Jianhua Yu, and Igor M. Samokhvalov, with several co-first authors (indicated by ‡). The study was primarily conducted by the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, City of Hope National Medical Center, Sun Yat-Sen University, Lomonosov Moscow State University, and other renowned international research institutions. The corresponding author is Dr. Igor M. Samokhvalov.

3. Study Workflow and Detailed Experimental Design

1. Overall Experimental Design

The entire study centers on “establishing RUNX1 reporter and knockout human pluripotent stem cell (hPSC) models via gene editing, and systematically tracking RUNX1 expression and function in an in vitro system simulating early human hematopoietic development”. The experimental workflow consists of the following major components:

• a) Construction of RUNX1 Reporter/Knockout hPSC Cell Lines:
  TALEN (Transcription Activator-Like Effector Nucleases) technology was used to integrate a fluorescent protein (tdTomato or Venus) reporter cassette into the RUNX1 gene (specifically in exon 3), achieving functional disruption of the RUNX1 gene via homologous recombination. The accuracy of targeted integration was validated by PCR and Southern blotting, yielding monoallelic and biallelic knockout cell lines.

• b) hPSC Differentiation to Simulate Human Hematopoietic Development System:
  A cytokine-free, highly reproducible embryoid body (EB)–based differentiation system was used to mimic early human hematopoietic development, with dynamic sampling of cells at distinct differentiation stages. Subpopulations of hematopoietic and non-hematopoietic cells were phenotypically and functionally analyzed and purified via magnetic bead sorting and flow cytometry (FACS).

• c) Functional Detection at Molecular and Transcriptome Levels:
  A combined approach using real-time quantitative PCR (qRT–PCR), Western blotting, RNA sequencing (RNA–Seq), flow cytometry, and colony-forming unit (CFU) assay was employed; bioinformatics analyses utilized Bowtie2, RSEM, DESeq2, clusterProfiler, and other tools for large-scale data processing and pathway enrichment analysis.

• d) Systematic Assessment of the Impact of RUNX1 Loss on Developmental Fate:
  This included evaluation of primitive and definitive hematopoietic lineages, mesenchymal lineages, and vascular endothelial lineages, as well as transcriptomic tracing of key transcription factors and signaling pathways (such as BMP, SOX, etc.).

• e) RUNX1 Function Rescue Experiments:
  A doxycycline (Dox)–inducible PiggyBac-Runx1c transgenic system was constructed and transfected into biallelic RUNX1 knockout cells, to assess the ability of exogenous RUNX1 compensation to restore hematopoietic differentiation potential.

2. Experimental Subjects and Sample Size

• The primary experimental subject was the human embryonic stem cell line (H1 hESC). Multiple monoallelic and biallelic RUNX1 knockout reporter lines were generated via gene editing and screening, used in parallel with isogenic wild-type controls.
  
• For all main experimental groups (such as differentiation, transcriptomics, etc.), the sample size was typically three independent biological replicates; some experiments included two to four distinct genotypic isogenic cell subpopulations (e.g., KO-1 and KO-2 biallelic knockouts, Runx1^Tdt/+ monoallelic KO, wild-type).

3. Detailed Core Experimental Procedures

3.1 Establishment and Expression Tracking of RUNX1 Reporter and Knockout Cell Lines

• TALENs targeting the upstream region of RUNX1 exon 3 were designed; a homologous recombination strategy was used to efficiently insert a fluorescence-reporting transcription termination cassette and achieve precise knockout. Integration of the splicing acceptor from the Engrailed-2 gene enabled accurate mapping of RUNX1 promoter activation by the reporter.
• Flow cytometry and qPCR jointly showed that RUNX1 expression is activated at a very early stage of differentiation (day 2, in CD43^–CD235a^– progenitors), and widely present in undefined mesodermal cells, nascent blood cells, and hemogenic endothelium (HE). The expression intensity gradually decreased from hematopoietic induction to myeloid differentiation, reflecting its temporospatially specific regulatory process.

3.2 The Impact of RUNX1 Knockout on Primitive and Definitive Hematopoietic Development

• Primitive Hematopoiesis Blocked:
  Biallelic RUNX1 knockout cells could still generate CD34^+CD43^+ early hematopoietic progenitors (indicating that the initial hematopoietic transition is not absolutely dependent on RUNX1), but the proportion of more mature CD43-high expressing cells was significantly reduced. Primitive erythroid, megakaryocyte, and myeloid differentiation were all severely inhibited. CFU assays revealed that even monoallelic knockout resulted in decreased differentiation capacity (RUNX1 haploinsufficiency already impaired primitive hematopoiesis). Furthermore, key erythroid transcription factors such as GATA1, KLF1, and MYB were significantly downregulated.

• Definitive Hematopoiesis Completely Aborted:
  By day 12 of differentiation, biallelic RUNX1 knockout cells almost completely lost CD45^+ hematopoietic cell potential; the developmental system was entirely devoid of T lymphocyte and NK cell lineages, with total abrogation of hematopoietic clonogenic ability. Multiparametric FACS confirmed an abnormal expansion of HE cells, which failed to proceed through endothelial-to-hematopoietic transition (EHT) and downstream hematopoiesis.

3.3 Shift in Non-Hematopoietic Mesodermal Fate and Upstream Network Regulation

• RUNX1 deficiency led to the gradual expansion of CD43^–CD146^+CD90^+CD73^+ mesenchymal precursor cells (MPCs) and CD31^+CD34^+CD146^+ endothelial-like cells. Meanwhile, expression of key non-hematopoietic developmental genes—including members of the BMP pathway, SOX family transcription factors (particularly SOX9, SOX5/6), RUNX2, etc.—was upregulated, indicating enhanced MPC differentiation potential and a “directional switch” toward non-hematopoietic fates such as chondrogenesis and angiogenesis.
• Transcriptome analysis confirmed that differentially expressed genes (DEGs) during days 12–20 of differentiation were highly enriched in mesodermal non-hematopoietic developmental pathways (such as BMP signaling, SOX signaling, Notch pathway, cell adhesion molecules, etc.).

3.4 Exogenous RUNX1 Function Rescue Experiment

• Using the PiggyBac transposon system, a Dox-inducible RUNX1c expression cassette was transfected into biallelic RUNX1 knockout cells. Timely Dox addition at days 6–12 efficiently activated RUNX1 expression, markedly restoring the ability to generate CD45^+ and CD34^+CD45^+ hematopoietic cells. CFU assays confirmed synchronized rescue of erythroid, myeloid, and megakaryocyte colony-forming potential, with these cells displaying strong mCherry/tdTomato double-fluorescence labelling.
• May-Grünwald staining further validated that the rescued cells possessed characteristic multi-lineage differentiation capacity, including typical megakaryocytes, macrophages, and granulocytes.

4. Main Research Results and Scientific Discoveries

1. RUNX1 is the top-level inducer of human hematopoiesis, essential for the initiation and progression of both primitive and definitive hematopoietic waves.
   RUNX1 deficiency blocks differentiation from HE to all mature hematopoietic cell types, proving its upstream role exceeds that of classical regulatory pathways such as TAL1/SCL in mice. RUNX1 is indispensable for the expression of key human hematopoietic transcription factors (including SCL/TAL1, GATA2, GFI1/1B, FLI1, etc.).

2. RUNX1 uniquely regulates human mesodermal multipotency and directly suppresses non-hematopoietic lineage expansion.
   In hematopoietic differentiation, RUNX1-deficient cell lines exhibited abnormal expansion and functional differentiation of non-hematopoietic cells, accompanied by upregulation of essential BMP–SOX pathway signals and dramatic suppression of hematopoiesis-related genes. This expands the known range of RUNX1’s impacts to bone, cartilage, vascular development, and EMT (epithelial–mesenchymal transition).

3. First revelation of a RUNX1-independent primitive erythroid cell subpopulation in human hematopoietic development.
   This subset of CD235^highCD34^–CD41a^– primitive erythroid cells arises early during differentiation, is RUNX1-independent, and lacks clonogenic potential, suggesting the existence of a special differentiation path distinct from classical hematopoietic stem/progenitor cells.

4. RUNX1 haploinsufficiency and precise control of hematopoietic homeostasis.
   Monoallelic knockout exhibited “insufficient dosage” phenotype with partial reduction in hematopoietic capacity, reflecting the critical threshold of RUNX1 dosage in hematopoietic system development.

5. Exogenous functional compensation can precisely restore hematopoietic ability, showing clinical potential.
   Inducible RUNX1c complementation effectively rescued multi-lineage hematopoietic function, providing direct experimental evidence for stem cell therapy, genetic correction, and AML-related technologies.

5. Conclusions, Significance, and Application Value

This study clearly proposes and rigorously demonstrates that: RUNX1 is not only the “master switch” for early human hematopoietic induction, but also actively and continuously regulates mesodermal lineage branching—activating programs for hematopoietic differentiation while synchronously suppressing excessive expansion of non-hematopoietic mesenchymal/endothelial fate. These findings not only reshape the conceptual framework of human hematopoietic regulation but also profoundly reveal the fundamental importance of balance between hematopoietic and non-hematopoietic development for embryogenesis and stem cell differentiation.

With respect to understanding pathogenesis in AML and other leukemias, bone marrow microenvironmental defects, and dysfunctionality of non-hematopoietic lineages such as MSCs in hematologic diseases, this study provides a robust theoretical and experimental foundation. Importantly, its comprehensive “induction–reporting–knockout–function rescue” research chain and innovative cell line resources pave the way for new directions in regenerative medicine, stem cell therapy, and developmental biology.

6. Research Highlights and Innovations

• Innovative Experimental Models: For the first time, a high-throughput, visual-tracing, and knockout-integrated RUNX1 loss-of-function hPSC cell line was established, constructing a high-resolution cellular fate mapping system.
• Multi-omics Integrated Analysis: The research covers phenotype, clonogenicity, molecular, and transcriptomic levels, thoroughly detailing the genetic regulatory network and verifying functional relevance.
• Deep Investigation into Non-Hematopoietic Fate Control Mechanisms: Breaking the traditional paradigm of hematopoietic-focused research, the study systematically elucidates the molecular basis for RUNX1 in regulating mesodermal developmental diversity, offering major insights for embryonic developmental balance and disease pathogenesis.
• Outstanding Clinical Translation Prospect: The rescue experiments demonstrate the precise, RUNX1-dependent regulation of directed differentiation in pluripotent stem cells, providing new strategies for hematopoietic reconstruction, gene therapy, and prevention and treatment of hematologic diseases.

7. Other Valuable Information

• Research data have been publicly released in the GEO database (accession GSE232147), offering a foundational resource for further analysis and translational research;
• The paper’s appendix supplies detailed information on reagents, experimental protocols, raw data, and data analysis code, ensuring reproducibility;
• The author team comprises leading basic and translational medicine groups from China, the USA, and Russia, illustrating a powerful example of international collaboration in tackling major frontier questions in developmental biology.

8. Overall Assessment and Prospects

This study not only puts forward a novel theory of the regulation of human hematopoietic fate and balance in mesodermal development, but also brings broad prospects for stem cell development and disease therapy involving hematopoietic, skeletal, vascular, cartilage, and other systems. In the future, such “multi-potency regulation–multi-lineage balance” mechanisms are expected to become new entry points for studying other developmental decisions and chronic disease pathogenesis. In-depth exploration of RUNX1 and its network mechanisms will continue to drive theoretical and applied innovations in biomedicine, stem cell research, and regenerative medicine.