Overexpression of ZIC1 Transcription Factor in Segmental Bone Defects Promotes Brown Adipogenic and Osteogenic Differentiation

Bioengineering Basic Medical Sciences Cell Biology Biochemistry Life Sciences Medicine Orthopedics
ZIC1 transcription factor osteogenic differentiation brown adipogenesis segmental bone defect stem cells human adipose progenitor cells hedgehog signaling

1. Academic Background and Research Significance

Human bone tissue possesses a certain degree of self-healing capability. However, after suffering from severe trauma, tumor resection, infection, or congenital deformity, leading to large-volume bone defects, this regenerative ability becomes limited. The so-called “critical size bone defect” refers to bone defects that are unable to heal spontaneously under natural conditions. Such issues are not only a challenge in clinical treatment but also impose a tremendous social and economic burden. The current mainstream treatments, such as autologous bone grafting, distraction osteogenesis, and allogenic bone material implantation, are plagued by multiple surgeries, long rehabilitation periods, and limited sources of graft material. Therefore, the development of new, safe, and effective bone regeneration technologies has become a common goal in the fields of tissue engineering and regenerative medicine.

In recent years, mesenchymal stem/progenitor cells (MSCs), because of their strong multilineage differentiation potential and accessibility, have become important candidate cells for bone tissue repair. In particular, human adipose-derived stem/stromal cells (hASCs), which can be easily obtained in large quantities, have garnered widespread attention and achieved certain success in bone repair for cranio-maxillofacial defects. However, for critical size bone defects and weight-bearing bones such as the femoral segment model, the reparative effect of hASCs has been less than satisfactory, with common problems including insufficient osteogenic capability and heterogeneous cell subpopulations.

The regulatory mechanisms governing stem cell differentiation fate are a hot topic in the field. Multiple studies have confirmed that transcription factors (TFs) are core regulators controlling MSCs’ differentiation towards osteogenesis or adipogenesis. For example, Runx2/Osterix promote osteogenic differentiation, whereas Pparγ/Cebps promote adipogenesis. Recently, Zic1 (Zic family member 1), a C2H2-type zinc finger transcription factor, has been identified as an important regulator of brown adipogenesis and skeletal development, and is proven under certain conditions to promote osteogenic differentiation and inhibit white adipogenesis. Previous in vitro studies have shown that Zic1 overexpression, via the Hedgehog signaling pathway, biases differentiation towards osteogenesis and affects the bone-fat balance, providing a theoretical foundation for enhancing the osteogenic potential of adipose-derived progenitor cells through gene regulation.

This study aims to address the bottleneck of inadequate efficacy in repairing large-volume bone defects with adipose-derived progenitor cells. It explores guiding adipose-derived stem cells towards preferential osteogenesis and suppressed adipogenesis through overexpression of the transcription factor Zic1, thereby improving the repair efficiency of large bone defects, while investigating the underlying molecular mechanisms and relevant signaling pathways.

2. Paper Source and Research Team Profile

This publication is an original experimental study entitled “zic1 transcription factor overexpression in segmental bone defects is associated with brown adipogenic and osteogenic differentiation”, published in the international top-tier journal Stem Cells in June 2025. The research was jointly conducted by team members including Neelima Thottappillil, Zhao Li, Xin Xing, etc., with the primary authors and team affiliated with the Department of Pathology at Johns Hopkins University, UCLA School of Dentistry, among other institutions. The project leader is Dr. Aaron W. James (corresponding author). The research was funded by the NIH, U.S. Department of Defense, American Cancer Society, and several other agencies and represents the international frontier in stem cell-based bone repair and differentiation mechanism research.

3. Research Design and Experimental Workflow Details

1. Overall Research Design

This study focuses on the function of the Zic1 transcription factor. By performing genetic manipulation to overexpress Zic1 in human adipose-derived stem/progenitor cells (hASCs), it systematically investigates its effect on osteogenesis and brown adipogenic differentiation and validates its function in vivo using animal models of large bone defects. The overall research flow includes:

  • Isolation, culture, and genetic modification (Zic1 overexpression) of hASCs
  • In vitro osteogenesis and brown adipogenic phenotype characterization and molecular mechanism analysis
  • In vitro and in vivo ectopic bone formation assay to confirm osteogenic potential
  • In vivo transplantation using a murine critical-size femoral segmental defect model
  • Multi-dimensional assessment of osteogenesis and differentiation characteristics by micro-CT, histology, immunofluorescence, etc.
  • Analysis of key signaling pathways (Hedgehog) and expression of brown adipogenic lineage marker molecules

Each experimental step is intricately linked, revealing both in vitro phenotypes and molecular mechanisms, and integrates in vivo regenerative validation with molecular functionality, thus exhibiting strong systematic and innovative features.

2. Experimental Subjects and Detailed Procedures

(1) Stem Cell Isolation and Zic1 Genetic Engineering

  • Source: Adipose tissue from healthy adult volunteers (obtained via liposuction surgery), following IRB approval (Johns Hopkins University IRB #0011905).
  • Isolation: Fat was washed with PBS, digested with 1% type II collagenase, and centrifuged to obtain the stromal vascular fraction (SVF). This was subjected to red blood cell lysis and filtered through a 40 µm sieve, yielding hASCs, which were then cultured in DMEM with 10% FBS and 1% antibiotics at 37°C, 5% CO₂.
  • Genetic manipulation: Mammalian expression plasmids containing the human Zic1 open reading frame (ORF) (Origene) were used for transfection with the Mirus Transit-X2 system. At 80% confluence, 1 μg of plasmid was used per well, with empty vector as control. RNA was extracted 48 hours post-transfection, with qRT-PCR used to confirm Zic1 overexpression.

(2) In Vitro Analysis of Osteogenic and Brown Adipogenic Differentiation

  • Osteogenesis Induction: Following transfection, cells were seeded into an osteogenesis induction medium (containing 10 mM β-glycerophosphate, 50 μM ascorbic acid, 1 mM dexamethasone) for 14 days, with media changed every other day. Osteogenesis was assessed via Alizarin Red S staining for calcium nodule deposition and quantitated via spectrophotometry (A584nm).
  • Molecular Analysis: qRT-PCR was used to detect expression of osteogenic genes (ALPL, Osterix/SP7, Osteocalcin/OCN) and brown adipogenesis genes (UCP1, CIDEA), compared to empty vector controls.
  • Brown Adipogenic Protein Detection: Immunofluorescent co-staining detected classic brown adipocyte markers such as Early B Cell Factor 2 (EBF2).

(3) In Vitro and Ectopic Bone Animal Models

  • Ectopic Ossicle Formation: 3×10⁶/ml transfected cells were mixed with hydroxyapatite/β-tricalcium phosphate (HA/β-TCP, 6:4), incubated at 37°C for cell adherence, and then subcutaneously implanted into the dorsal surface of NOD-SCIDγ mice. Each mouse received the control group on one side and the Zic1 overexpression group on the other (n=6). Samples were harvested after 12 weeks for detection.
  • Femoral Segmental Bone Defect Model: A 3.5 mm-long defect was created in the distal femur of NOD-SCIDγ mice. Custom HA/PLGA cylindrical scaffolds (3.5 mm × 2 mm) were seeded with 8×10⁵ cells (Zic1 OE or LV control), incubated at 37°C for 15 minutes, and implanted into the defect site, fixed with P.E.E.K. micro-locking plates. Tissues were harvested for analysis 8 weeks post-surgery (n=4).

(4) Multi-omics Detection and Data Analysis

  • Micro-CT: At 12 weeks (ectopic) and 8 weeks (segmental defect), samples were reconstructed in 3D and quantitatively measured for bone density, volume, and trabecular thickness. The ROI was the 4.5 mm × 2.5 mm region within the scaffold. Analysis utilized NRecon, CTAn software, etc.
  • Histology and Immunohistochemistry: Samples were decalcified, OCT-embedded, and cryosectioned (15 μm), followed by H&E, Goldner trichrome, and Picrosirius Red staining. Optical and polarized light microscopy was used to quantitatively assess bone area and collagen fiber density. Immunofluorescence was used to detect human/mouse nuclei, Zic1, Ocn, Runx2, Ptch1, EBF2, and vascular markers CD31, Endomucin, etc., with multi-color co-staining and image analysis using ImageJ/Imaris.
  • Statistical Analysis: All experiments were performed in triplicate, with data presented as mean ± standard deviation (SD). Student’s t-test and ANOVA (Tukey’s multiple comparison) were used for statistical analysis, with p<0.05 as the cutoff for significance.

4. Main Experimental Results and Findings

1. Zic1 Overexpression Enhances hASC Osteogenesis and Ectopic Bone Formation

  • qRT-PCR showed a 115% increase in Zic1 gene expression 48 hours post-transfection; at 10 days of osteogenic induction, the Zic1 OE group had a 32% higher calcium nodule absorbance than controls.
  • Osteogenic gene expression increased (ALPL up 70%, OCN up 90%, Osterix up 40%).
  • Micro-CT in the ectopic bone model revealed that bone volume fraction (BV/TV) in Zic1 OE group increased by 47% versus controls; immunohistochemistry showed a significant increase in Zic1-positive cells (86%); Ptch1 (sonic hedgehog response gene) expression increased by 280%, indicating osteogenic differentiation was related to enhanced Hedgehog signaling.

2. Zic1 Overexpression Promotes Osteogenic Differentiation in Large Bone Defects but Does Not Achieve Bony Union

  • 3D-reconstructed micro-CT of femoral segmental defects showed that the Zic1 OE group exhibited significant new bone formation at the defect perimeter, but no central bony bridging; overall BV and BV/TV were increased by 46% and 42%, respectively, with greater trabecular thickness.
  • H&E, Goldner trichrome, and Picrosirius Red quantitative analyses all confirmed that new bone area increased by 65% in the Zic1 OE group compared to controls, along with higher collagen density, indicating enhanced extracellular bone matrix formation although the structure was not continuous.

3. Osteogenic Differentiation, Hedgehog Signaling, and Human Cell Persistence In Vivo

  • Human cell retention in bone defects (HNA marker) was similar in both groups, but the Zic1 OE group showed increased Ocn and Runx2 protein expression (by 80% and 89%, respectively), corresponding to the new bone formation phenotype.
  • Analysis of the Hedgehog signaling pathway demonstrated that expression of Zic1 and Ptch1 in the OE group increased by 1800% and 261%, respectively, both co-localizing with human cells, suggesting that Zic1 promotes osteogenesis via enhancement of Hedgehog signaling.

4. Zic1 Promotes hASC Brown Adipogenic Differentiation Tendency

  • qRT-PCR showed UCP1 and CIDEA (brown adipogenic markers) expression increased 15–19 fold in the Zic1 OE group under both routine and osteogenic conditions, indicating a markedly activated brown adipogenic phenotype.
  • EBF2 protein expression in bone defect tissue increased by 300%, co-localized with human cells, but no mature adipocyte formation was observed histologically, suggesting an early brown adipogenic differentiation status.
  • Angiogenesis analysis (CD31, Endomucin) found no significant difference between groups, suggesting that enhanced osteogenic and brown adipogenic differentiation was not dependent on increased vascularization.

5. Research Conclusion and Scientific Value

This study is the first to systematically demonstrate that genetic engineering to overexpress the Zic1 transcription factor effectively enhances the osteogenic and brown adipogenic differentiation of human adipose-derived mesenchymal progenitor cells, and promotes new bone formation in a critical size bone defect animal model. Although full bony bridging was not achieved, there was significant improvement in osteogenesis, extracellular matrix remodeling, and activation of bone-related signaling pathways.

1. Scientific Significance

  • The study elucidates the molecular mechanism by which mesenchymal progenitor cell fate is regulated by the transcription factor Zic1, particularly in balancing osteogenesis/brown adipogenesis and coupling to Hedgehog signaling;
  • It demonstrates that although Zic1 regulation alone can enhance osteogenic potential, complex tissue regeneration still requires combinatorial strategies, thus providing a theoretical and methodological basis for multi-target stem cell-based bone repair;
  • It reinforces the potential application of adipose-derived stem cells for critical size bone defect therapy, advancing towards clinical translation.

2. Application Prospects

  • Genetic engineering optimization of seed cells’ osteogenic capacity offers new cell-therapy avenues for patients with refractory large-volume bone defects;
  • Development of drugs targeting Zic1 or Hedgehog signaling provides new molecular targets for enhancing bone repair efficacy;
  • Combining with traditional tissue engineering, scaffold materials, vascularization/innervation technologies, lays the foundation for the design of multi-factor combinatorial treatment approaches.

6. Research Highlights and Innovations

  • Innovative Research Paradigm: Deeply integrates genetic regulation (Zic1 overexpression) with the bone repair function of pediatric/adult adipose-derived mesenchymal cells, rigorously validating through comprehensive in vitro phenotype and animal models;
  • Mechanistic Insights: Clarifies the pivotal role of Hedgehog signaling in Zic1-mediated regulation of hASC osteogenic/brown adipogenic differentiation, verifying the upstream transcription factor–downstream signaling axis;
  • Technical Approach: Multi-process, multi-level (molecule–cell–tissue–animal) integrated analysis, comprehensively covering genetic engineering, osteogenic induction, animal modeling, 3D imaging, immuno- and collagen analysis using cutting-edge techniques;
  • Unique Research Object: Focuses on critical-size bone defects and the high-application-potential seed cell type—adipose-derived cells;
  • Theory and Clinical Practice Integration: Addresses both fundamental mechanism and real-world application bottlenecks (e.g., incomplete bony union), facilitating sustainable clinical advancement.

7. Other Valuable Information

  • The research team is multidisciplinary, combining expertise from pathology, engineering, surgery, etc., demonstrating a high degree of cross-innovation.
  • Data and materials are openly shared, available in the Supplementary Material and from the corresponding author, facilitating community re-analysis and follow-up studies.
  • Principal investigator Aaron W. James has extensive experience in stem cell and bone tissue engineering, ensuring scientific rigor and regulatory compliance.
  • Research funding is supported by NIH, the U.S. Department of Defense, etc., with project design closely related to clinical translation.

8. Conclusion

This study provides a new biological foundation and regulatory strategy for bone tissue engineering, and also indicates that functional repair of critical-size bone defects still requires multi-dimensional optimization. Genetic regulation of transcription factors such as Zic1 demonstrates great potential for precise modulation of stem cell differentiation and enhancement of bone repair capacity. Looking forward, further integration with materials science, pharmacology, and regenerative microenvironments may offer systematic solutions to the challenges of bone regeneration, bringing new hope for patients.