Rabbit Induced Pluripotent Stem Cells-Derived Mesenchymal Stem Cells for Enhanced Wound Healing

Bioengineering Basic Medical Sciences Cell Biology Life Sciences Medicine Orthopedics
induced pluripotent stem cells mesenchymal stem cells rabbit wound healing regenerative medicine SB431542 differentiation

Academic Background and Research Motivation

In recent years, the field of stem cell and regenerative medicine has developed rapidly. Due to their multipotency and self-renewal capacity, stem cells have become a vital cell source for tissue repair and regeneration. Among the many types of stem cells, Mesenchymal Stem Cells (MSCs) have drawn considerable attention because of their broad tissue repair abilities demonstrated in both animal and clinical studies. MSCs can be derived from various tissues such as bone marrow, adipose tissue, dental pulp, synovium, and umbilical cord from different animals or sources[2][3][4][5][6][7]. However, the clinical application of MSCs is limited by two major challenges: first, it is difficult to isolate and expand sufficient numbers of high-quality MSCs in vitro, especially as the donor ages, the number and functionality of MSCs further decline[8]; second, prolonged in vitro culture leads to senescence, reduced differentiation capacity, and functional loss in MSCs[9].

To address the problems of MSC source and functional decline, the emergence of Induced Pluripotent Stem Cells (iPSCs) has brought new hope. iPSCs have extremely high proliferation and multipotency, and through reprogramming, they can eliminate aging-associated phenotypic barriers such as telomere shortening, mitochondrial dysfunction, and cell cycle arrest[11][12]. In theory, iPSCs can efficiently generate MSCs through specific differentiation pathways, thus providing abundant high-standard cell resources for clinical and regenerative medicine applications[10][15]. Although differentiation studies on human and murine iPSCs are relatively mature, there is still a lack of in-depth and systematic research on the mechanisms, characteristics, and application models for iPSCs and their derived MSCs in other species (especially large animals such as rabbits). This directly impacts animal model selection, the development of veterinary clinical treatments, and the advancement of cross-species regenerative medicine.

This study is carried out in this context, aiming to explore feasible approaches for efficiently differentiating rabbit induced pluripotent stem cells (rabbit iPSCs) into mesenchymal stem cells (MSCs), to establish a reliable in vitro induction protocol, and to verify the wound healing promoting function of the obtained iPSCs-MSCs in a mouse injury model, thus providing a theoretical basis and experimental evidence for cross-species applications in regenerative medicine.

Source of Paper, Research Team, and Publication Information

This paper, titled “Rabbit induced pluripotent stem cells-derived mesenchymal stem cells for enhanced wound healing,” was completed by Hsing-Yi Yu, Yang-Zhe Huang, Edward Chern, et al. The authors are affiliated with the Niche Lab for Stem Cell and Regenerative Medicine, Department of Biochemical Science and Technology, and the Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University. The article was published in 2025 in the journal Stem Cells Translational Medicine (stmcls) under Oxford University Press.

Research Design and Detailed Experimental Procedures

Overview of the General Experimental Structure

The paper presents a comprehensive experimental design focused on the efficient differentiation of rabbit induced pluripotent stem cells into mesenchymal stem cells. The main experimental workflow consists of: a) rabbit iPSCs culture and differentiation; b) molecular characterization and pluripotency assessment of differentiated cells; c) testing the trilineage differentiation potential of iPSCs-MSCs; d) in vivo functional validation of iPSCs-MSCs in a mouse wound healing model, supporting the study’s conclusions with a series of data.

1. Culture and Passaging of Rabbit Induced Pluripotent Stem Cells

The rabbit iPSC line (ips-L1) used in this study was provided by RIKEN BioResource Center, Japan. The cells were cultured using standard KOSR medium (containing 20% knockout serum replacement, bFGF, GlutaMAX, non-essential amino acids, and β-mercaptoethanol) on plates pre-seeded with mouse embryonic fibroblasts (MEFs) as feeder layers. When iPSCs reached 50-60% confluence, they were passaged using low-concentration trypsin at appropriate densities.

2. Detailed Procedure for Differentiation of Rabbit iPSCs into MSCs

Differentiation utilized the embryoid body (EB) formation method combined with the TGF-β signaling inhibitor SB431542.

  • Step 1: After enzymatic digestion, rabbit iPSCs were resuspended and cultured in non-adherent 6-well plates (about 1.5×10^5 cells/well) to form spherical embryoid bodies. EBs were cultured in KOSR medium for 10 days, with medium changes at regular intervals.
  • Step 2: Embryoid bodies were transferred to gelatin-coated culture dishes with the addition of 10 μM SB431542 to promote differentiation towards the mesenchymal lineage, with medium changes every 3 days.
  • Step 3: After 20 days, outgrowth cells from EBs were trypsinized and transferred to 60mm dishes for further culture, a process in which SB431542 was continually added to the medium to form iPSCs-MSCs.
  • Step 4: After the first passage, when cells reached 80% confluence they were passaged at a 1:3 ratio. The entire culture process lasted about 27 days, ultimately yielding rabbit iPSCs-MSCs that met the morphological, genetic, and functional criteria for MSCs.

3. Molecular and Functional Characterization of Differentiated Cells

  • Loss of Pluripotency and Germ Layer Differentiation: RT-PCR and qPCR were used to confirm the progressive silencing of pluripotent genes such as Oct4, Sox2, and Nanog during differentiation; the expression changes of three germ layer markers in EBs and early differentiated cells provided evidence for lineage specification.
  • Surface Marker Detection: Expression of MSC-specific surface markers (CD44, CD73, CD90, CD105) was assessed by qPCR to demonstrate that the differentiated cells acquired typical MSC phenotypes; at the same time, the hematopoietic stem cell markers CD34 and CD45 were completely undetectable, indicating high cell purity.
  • Comparison with Rabbit Adipose-Derived MSCs (ADSC): iPSCs-MSCs showed higher levels of MSC-specific marker expression than rabbit adipose-derived MSCs, demonstrating the effectiveness of the differentiation protocol.

4. In Vitro Multilineage Differentiation Potential Assessment

The osteogenic, adipogenic, and chondrogenic differentiation potentials were tested:

  • Osteogenic Differentiation: After exposure to osteogenic induction medium, Alizarin Red S staining was used to detect calcium deposition, and qPCR was performed for osteogenic genes such as ALP and Runx2, confirming osteogenic potential.
  • Adipogenic Differentiation: After transfer to adipogenic induction medium, Oil Red O staining was used to detect lipid droplets, with the expression of adipocyte differentiation genes FABP4 and PPARγ indicating strong adipogenic potential.
  • Chondrogenic Differentiation: High-density micromass culture was used. Alcian blue staining assessed the accumulation of hyaluronic acid and proteoglycan, while the significant upregulation of chondrogenic genes Col2A1, ColXA1, Sox9, and Acan was demonstrated.

5. In Vivo Functional Test of Rabbit iPSCs-MSCs in Promoting Mouse Wound Repair

A full-thickness skin excision model (5 mm) in immunodeficient nude mice was used for validation. The experimental group received local injections of Matrigel-embedded iPSCs-MSCs in the wound area, with additional ring-shaped peripheral injections of MSC suspension; the control group received only PBS. Wound size was photographed and measured on days 0, 3, 7, and 10, and area quantification was done using ImageJ. The results showed that the iPSCs-MSCs group significantly accelerated wound healing compared to controls on both days 3 and 10 (P<0.05). The animal experiments were approved according to international ethical standards.

Key Experimental Results and Data Support

  1. Rabbit iPSCs are capable of EB formation and trilineage differentiation potential. Using the SB431542 induction protocol, they efficiently differentiate towards the mesenchymal lineage, with significant upregulation of mesenchymal markers such as BMP4 and downregulation of ectodermal and endodermal markers.
  2. The iPSCs-MSCs obtained from the protocol not only resemble conventional ADSCs in surface marker expression profile but also show dynamic gene expression curves of positive and negative markers, indicating that this induction protocol effectively eliminates residual pluripotent stem cells and purifies the MSC lineage.
  3. iPSCs-MSCs have outstanding in vitro trilineage differentiation capability, with relevant functional gene expression elevated by tens to thousands of times compared to controls in each differentiation type, and tissue staining demonstrating clear differentiation.
  4. In the wound repair animal experiment, the iPSCs-MSCs transplantation group showed superior wound closure speed and healing quality compared to controls, validating actual in vivo functionality. Regarding safety, there was no abnormal proliferation or tumor formation at the transplantation sites within two months, supporting the biological safety of these cells.

Research Conclusion, Scientific and Application Value

This study has established a complete, efficient, and reliable protocol for the differentiation of rabbit iPSCs into functional MSCs, fully validating the high universality and efficiency of TGF-β signaling inhibitor (SB431542) in directed induction of MSCs across species. The obtained iPSCs-MSCs exhibited classical trilineage differentiation capacities in vitro and demonstrated significant wound healing enhancement in the mouse injury model. By comparing different animal models, the paper further argues that rabbits are uniquely advantageous as models close to humans for regenerative medicine and disease research, laying a strong foundation for future stem cell therapies and personalized medicine.

In addition, concerning the potential tumorigenicity risk of cell therapy, the authors observed in vivo safety for up to two months after transplantation, with no tumor formation at injection sites, thus providing new reference data for preclinical safety evaluation of cell transplantation.

Research Highlights and Innovations

  • A novel and efficient differentiation protocol combining EB formation and SB431542 induction for rabbit iPSCs-MSCs was established and optimized, providing a new template for directed MSC induction from PSCs in different species.
  • For the first time, the molecular profile, functional characteristics, and performance advantages of rabbit iPSCs-derived MSCs compared with conventional ADSCs were systematically elucidated, enriching comparative biological evidence of interspecies differences in stem cell biology.
  • Employing rabbit iPSCs-derived MSCs in a cross-species (rabbit→mouse) wound healing model was a breakthrough, strongly supporting the efficacy and safety of MSCs in xenogeneic tissue repair and laying important experimental and theoretical groundwork for large animal and even clinical translation.
  • A novel finding that rabbit iPSCs are more sensitive to SB431542 and tend towards adipogenic differentiation during MSC induction broadens our understanding of TGF-β signaling regulation in the differentiation mechanisms of pluripotent stem cells.

Limitations and Outlook

The authors frankly acknowledge that the current wound repair experiments used rabbit iPSCs-MSCs in a xenogeneic (rabbit-mouse) setting, and further validation should extend to autologous or allogeneic animal models to more accurately assess their value as clinical simulators. Furthermore, future research will explore the molecular interactions between iPSCs-MSCs and the injury microenvironment, as well as their long-term safety and applications in chronic disease models in vivo.

Summary and Significance

This study has achieved a systematic breakthrough in the field of induction and functional application of mesenchymal stem cells in animals, providing not only technical support for advanced animal models in stem cell therapy and veterinary clinical applications but also paving a new way for large-scale production, matching, and personalization of human iPSCs-MSCs in regenerative medicine. The paper sets a high standard for analytical procedures and multi-level data support, with rigorous content and clear logic, thus offering important theoretical and technical guidance for advances in regenerative medicine, animal model research, and stem cell therapies.