Research ArticleExperimental Studies
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Preclinical Assessment of Safety and Efficacy of Deferoxamine (DFO)-pretreated Feline Adipose Mesenchymal Stem Cells

HAILONG TENG, MINKYUNG CHO, ZHIQIANG MA, XINPENG JI, SHIYU JIN, YUZE ZHOU, MEIFU XUAN, HYUN-A SEONG, XIANGZI LI, SEONG-HO CHOI and QIANG LI
In Vivo January 2025, 39 (1) 267-279; DOI: https://doi.org/10.21873/invivo.13825

Abstract

Background/Aim: This study aimed to investigate the safety and efficacy of deferoxamine (DFO) pretreated feline adipose tissue derived mesenchymal stem cells (fATMSCs) for the treatment of inflammatory disorders. Materials and Methods: fATMSCs were isolated from feline adipose tissue and characterized using flow cytometry for surface marker expression and differentiation assays for adipogenic, osteogenic, and chondrogenic lineages. Different concentrations of DFO were used to evaluate its impact on fATMSC activity. The therapeutic potential of these preconditioned cells was validated using a mouse model of acute lung injury (ALI) by LPS injection. Comprehensive evaluations, including clinical, hematological, and radiological assessments, were conducted before and after intravenous injection of preconditioned cells in three feline subjects. Results: 25 μM DFO pretreatment significantly up-regulated immunomodulatory genes (Tgfb, Hgf, and Tsg-6) in fATMSCs. In the mouse ALI model, DFO-pretreated fATMSCs exhibited enhanced anti-inflammatory effects, reducing inflammatory cytokines (Tnfa, Il1b, Il6). Clinical safety assessment in felines showed no immediate adverse effects, structural alterations, or tumorigenesis. Conclusion: Utilizing a mouse model of acute lung injury, we demonstrated the potential of DFO-pretreated fATMSCs as a safe and effective therapeutic approach for inflammatory disorders.

Key Words:

In veterinary medicine, the application of stem cell therapy has garnered significant interest, particularly for treating inflammatory conditions in companion animals (1-3). Among the diverse sources of stem cells, adipose tissue-derived mesenchymal stem cells (ATMSCs) have become a promising candidate due to their ease of extraction, high yield, and potent regenerative capabilities (4). In particular, feline ATMSCs (fATMSCs) present unique therapeutic potential for managing feline-specific conditions, such as chronic kidney disease and inflammatory disorders (5). However, despite these promising applications, there are persistent concerns about the safety and efficacy of stem cell-based therapies in veterinary settings, especially concerning allogeneic transplantation and long-term effects (6).

Consequently, current research endeavors are focused on developing new strategies based on previous findings aimed at enhancing the safety and efficacy of stem cells (7-9). The objective is to support the advancement of more sophisticated stem cell therapies. Recent studies have concentrated on optimizing stem cell preconditioning methods to enhance both the safety and therapeutic efficacy of MSCs. Acute lung injury (ALI), a severe syndrome with high mortality, is characterized by acute respiratory failure and accompanied by damage to lung cells, leading to inflammation, hemorrhage, and edema (10, 11). The lipopolysaccharide (LPS)-induced ALI model is widely used due to its reproducibility (12). This model induces lung inflammation and increases vascular permeability, causing symptoms like pulmonary edema. Pathological assessment is crucial for evaluating lung damage. Currently, effective treatments for ALI are lacking. Therefore, in this study we innovatively used deferoxamine (DFO) to pretreat adipose tissue-derived mesenchymal stem cells (ATMSCs) based on previous research findings, exploring a new therapeutic strategy (13).

Deferoxamine (DFO), an iron-chelating agent, has demonstrated potential in preconditioning stem cells to improve their survival, engraftment, and therapeutic outcomes (14). Additionally, investigations into the hypoxic preconditioning of MSCs have revealed that DFO serves as a potent inhibitor of prolyl hydroxylase, widely used to stabilize hypoxia-inducible factor (HIF-1α) in normoxic conditions (15, 16). This attribute is particularly beneficial for adipose-derived MSCs, as the hypoxic environment induced by DFO promotes cell differentiation, survivability, and significantly enhances angiogenesis by modulating the expression of angiogenic factors (17, 18).

The benefits of DFO pretreatment are well-established, and the high biosafety and aqueous solubility of DFO have made it widely used in clinical practice (19). However, the safety of DFO-pretreated fATMSCs requires further investigation. Before DFO-pretreated fATMSCs are put into clinical practice, a rigorous and comprehensive preclinical safety assessment is necessary to confirm their safety and efficacy in veterinary medicine. This study systematically evaluated the effects of varying DFO concentrations on the biological activity of fATMSCs to identify the optimal concentration. The emphasis of this study was on investigating the efficacy and safety of DFO-pretreated fATMSCs using comprehensive in vivo experiments, as well as detailed hematological and radiological evaluations, to establish a fundamental understanding of the role of DFO in modulating the properties of fATMSCs.

Materials and Methods

Cell isolation and characterization. Adipose tissue was procured from the subcutaneous region of the abdomen of three feline subjects sourced from an animal healthcare facility, with explicit written consent obtained from the owners of the cats prior to their utilization in research. The procedure was approved by the Institutional Animal Care and Use Committee of Yanbian University and performed in accordance with approved guidelines (YD20240510059). The fATMSCs were extracted and cultured following established protocols (20). Subsequently, the cells underwent initial phenotyping to identify various recognized stem cell markers present on their surface using flow cytometry before being utilized in experimental procedures. Upon reaching the third generation, the cells were harvested through trypsin digestion to create a cell suspension containing 1×106 cells in 30 μl of phosphate buffer saline (PBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) solution, with the addition of 1 μl of monoclonal antibodies targeting specific proteins, namely CD9-fluorescein isothiocyanate (FITC) and CD44 (Invitrogen, San Diego, CA, USA), CD34- FITC, and CD45- phycoerythrin (PE; eBiosciences, San Diego, CA, USA). For CD44, indirect immunofluorescence was performed using anti-rat IgG-PE (Santa Cruz Biotechnology, Santa Cruz, CA, USA), conjugated with corresponding detection antibodies in separate test tubes. Unstained cells were employed as controls for autofluorescence. The cells were then analyzed using the BD FACS Aria II system (BD Bioscience). Subsequent cellular differentiation was confirmed by utilizing PRIME-XV® Chondrogenic Differentiation Xeno-Free Serum-Free Medium (XSFM), PRIME-XV® Osteogenic Differentiation Serum-Free Medium (SFM), and PRIME-XV® Adipogenic Differentiation SFM (all from Irvine Scientific, Santa Ana, CA, USA) in accordance with the manufacturer’s instructions, followed by staining with Alcian Blue, Alizarin Red, and Oil Red O for chondrogenic, osteogenic, and adipogenic differentiation, respectively.

Cell proliferation assay. To explore the impact of DFO on the proliferation of fATMSCs, various concentrations of DFO (25 μM, 50 μM, 100 μM, 200 μM, 400 μM and 800 μM) were administered to the cells, with PBS used as a control group. Following a 24-h incubation period, the viability of the cells was assessed through a Cell Counting-Kit (CCK-8) assay (Apexbio, Houston, TX, USA) and quantified by measuring the absorbance at 450 nm using a spectrophotometer (Bio-Rad Microplate Reader Model 680, Bio-Rad Laboratories, Hercules, CA, USA). The most effective concentration of DFO treatment was determined based on the analysis of cell viability. Subsequently, the impact of DFO at the optimal concentration on the proliferation of fATMSCs was further investigated through CCK-8 assays conducted at 24, 48, and 72 h.

Development of mouse model for ALI. Twenty male C57BL/6 mice, each weighing around 20-25 g, were procured from the Laboratory Animal Centre of Yanbian University. Following acquisition, a one-week acclimatization period was provided in the laboratory, maintaining a specific pathogen-free setting with controlled temperature (20-22°C), humidity (50%±5%), and a 12:12 light-dark cycle. The mice were categorized into four groups using the experimental animal interval grouping method: Naive group (group without any treatment), lipopolysaccharide (LPS)+PBS group, LPS+fATMSCs group, and LPS+DFO treated fATMSCs (LPS+DFO-fATMSCs) group. LPS-induced ALI was induced in the mice through an intraperitoneal (IP) injection of 10 mg/kg LPS from Escherichia coli (O55; Sigma-Aldrich). Subsequently, 3 h post-LPS injection, the mice in the LPS+PBS group received 100 μl of PBS via IP injection, while the mice in the LPS+fATMSC and LPS+DFO-fATMSCs groups were administered IP injections of 2×106 fATMSCs and fATMSCs preconditioned with DFO, respectively. Following a 24-h period after ALI induction, the mice were evaluated for abnormal behaviour using pathological scoring criteria (21).

RNA extraction and real-time quantitative PCR. Total RNA was isolated from lung tissues obtained from mice, as well as from fATMSCs cultured in vitro and DFO-pretreated fATMSCs, using Trizol Reagent (Invitrogen, Waltham, MA, USA). Subsequent cDNA synthesis was conducted using the FastKing one-step kit (Tiangen Biotech, Beijing, PR China). Gene expression analysis was performed using the SYBR premix SuperReal PreMix Plus Kit (Tiangen Biotech) with 400 nM of both forward and reverse primers listed in Table I. These procedures were carried out in an Agilent Mx3000/5p thermocycler (Agilent Technologies, Santa Clara, CA, USA). The expression levels of each gene were normalized to the glyceraldehyde 3-phosphate dehydrogenase (Gapdh) reference gene, and the relative gene expression was calculated relative to the control group.

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Table I.

Primer sequences for quantitative real-time polymerase chain reaction amplification of feline and mouse target genes.

Enzyme-linked immunosorbent assay (ELISA). ELISA was conducted to quantify the levels of TNF-α protein in the collected serum. From three cats, 3 ml of fresh blood was collected using vacuum needles and allowed to clot at room temperature for 1h. The samples were then centrifuged at 3,000 rpm for 15 min at 4°C, and the clear or pale-yellow upper layer was collected as the serum. The feline TNF-α ELISA kit (MyBioSource in San Diego, CA, USA) according to the manufacturer’s instructions. The procedure involved adding serum samples to wells coated with antibodies, followed by incubation, the addition of a detection antibody, and the introduction of a substrate for color development. Optical density readings were taken at 450nm to ascertain protein concentrations by comparing them to a standard curve.

Histopathological analyses. After euthanasia, the lung tissues were harvested for histopathological examination. To preserve cellular details and structural integrity, the tissues were initially fixed in a 10% formaldehyde solution. Subsequently, they were embedded in paraffin to aid in the sectioning process. For microscopic examination, 5-μm thick sections were prepared from the paraffin-embedded tissues. Each section was then stained with hematoxylin and eosin to enhance the visibility of cellular features and tissue architecture under the microscope.

Clinical trial procedures. To assess the safety of DFO-pretreated fATMSCs, three healthy cats were selected from those evaluated during a health assessment at Yanbian University Teaching Animal Hospital. Intravenous (IV) injections of DFO-pretreated fATMSCs at a dosage of 1×106 cells/kg were administered to the forelimbs of these cats. To mitigate cytotoxic effects and minimize the risk of thrombosis, the cells were suspended in 15 ml of PBS and delivered intravenously over a 15 min period using an infusion pump. Safety evaluations included physical examinations, with a focus on body temperature, respiratory rate, heart rate, and systolic blood pressure. These parameters were monitored one hour before and after the injection in all three cats, with data collection aimed at detecting adverse reactions and systemic inflammatory responses associated with cell therapy. Additionally, comprehensive ultrasound follow-up examinations were performed at 30 and 90 days post-injection to assess the long-term safety and potential carcinogenic effects of the injected fATMSCs.

Laboratory examinations. Blood samples were collected from the three felines for comprehensive hematological and biochemical analyses. The hematological parameters evaluated included white blood and red blood cell counts; hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, red blood cell volume distribution width-coefficient of variation; differential count of eosinophils, basophils, lymphocytes and monocytes; and platelet count. Serum biochemistry assessments included measurements of feline serum amyloid A, albumin, total proteins, globulin, albumin/globulin ratio, total bilirubin, aspartate aminotransferase, alanine aminotransferase, amylase, creatine kinase, creatinine, blood urea nitrogen, glucose and triglycerides.

Statistical analysis. All experimental data were analyzed using GraphPad Prism 9 (GraphPad Software Inc., La Jolla, CA, USA). One-way analysis of variance or Student’s t-test was used for between-group differences. Statistical significance was set at p<0.05.

Results

Characterization of fATMSCs. To assess the expression of surface antigens on the isolated cells for characterization, this study utilized flow cytometry. fATMSCs were successfully cultured and expanded to passage 3, with the majority of cells expressing established stem cell markers CD9 (99.13%) and CD44 (98.91%). Conversely, a minor proportion expressed CD34 (6.49%) and CD45 (5.73%) (Figure 1A). The fATMSCs demonstrated a capacity for multidirectional differentiation, as evidenced by their ability to undergo adipogenic, osteogenic, and chondrogenic differentiation. Following adipogenic induction, the fATMSCs displayed numerous bright red lipid droplets in their cytoplasm after Oil Red O staining. Subsequent induction of osteogenic differentiation resulted in abundant red calcified nodules within the cells, as revealed by Alizarin Red staining. Furthermore, chondrogenic differentiation induction revealed dark blue chondrogenic stromal cells through Alcian Blue staining, characterized by irregularly granular morphology (Figure 1B). In conclusion, these results confirm the successful induction of differentiation in fATMSCs, validating their potential for multidirectional differentiation.

Figure 1.
Figure 1.

Characterization of feline adipose tissue-derived mesenchymal stem cells (fATMSCs) based on surface marker expression and differentiation potential. (A) Flow cytometric histograms showing the expression of mesenchymal stem cell markers. (B) Potential of fATMSCs to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Magnification, 200×.

Effect of DFO on fATMSCs viability. To determine the effects of DFO on fATMSCs, we measured the proliferation rates of cells treated with various concentrations of DFO (0, 25, 50, 100, 200, 400, and 800 μM) after a 24-h incubation period. As shown in Figure 2A, following this treatment, the proliferative capacity of fATMSCs was significantly reduced at all concentrations of DFO, except for the 25 μM group. These results indicate that, aside from the 25 μM treatment group, the inhibitory effect of DFO on the proliferative activity of fATMSCs increased progressively with higher concentrations. Observation of cells treated with 25 μM DFO for 24, 48, and 72 h revealed no decrease in cell viability. Consequently, subsequent experiments employed a concentration of 25 μM DFO for treatment.

Figure 2.
Figure 2.

Evaluation of deferoxamine (DFO) preconditioning on feline adipose tissue-derived mesenchymal stem cells (fATMSCs). (A) Cell viability assay results showing the effects of different concentrations of DFO on fATMSCs over a 24 h period compared to untreated cells. Values with different letters are significantly different at p<0.05 according to one-way analysis of variance. (B) Comparison of the proliferative capacity of fATMSCs treated with 25 μM DFO versus untreated cells. (C) Quantitative real-time PCR analysis of interferon-γ (Ifnγ), transforming growth factor β (Tgfβ), hepatocyte growth factor (Hgf), and tumor necrosis factor α-induced protein 6 (Tsg6) mRNA expression in fATMSCs following DFO treatment and untreated (control) group. Data are expressed as the mean±standard deviation. ns: Not statistically significant. Significantly different at **p<0.01 versus the naive cells by Student’s t-test.

Effects of DFO on fATMSCs immunomodulation. To elucidate the immunomodulatory effects of DFO pretreatment on fATMSCs, we analyzed the gene expression levels of key immunomodulatory factors after 24 h of DFO treatment. The results demonstrated that DFO preconditioning significantly up-regulated the gene expression of Tsg6 and Hgf in fATMSCs (Figure 2C). However, no significant increase was observed in the expression of other immunoregulatory cytokines, such as Ifng and Tgfb.

Effect of DFO-fATMSCs on mouse model of ALI. In this study, we initially assessed the sick score of the mice and obtained their serum and lung tissues to investigate the therapeutic effects of DFO-preconditioned fATMSCs in a mouse model of ALI (Figure 3A) (22). Mice in the LPS+PBS group displayed severe symptoms, including wrinkled fur, perianal fecal staining, a curled body posture with minimal responsiveness to stimuli, and scaling around the eyes. In contrast, mice in the LPS+fATMSCs and LPS+DFO-fATMSCs groups exhibited significant improvements in their sick scores compared to the LPS+PBS group, with the LPS+DFO-fATMSCs group showing the most pronounced therapeutic effects (Figure 3B). Gene expression analysis indicated that, compared to the naïve group, the levels of Tnfα, Il1b, and Il6 in the lung tissue of the LPS+PBS group were significantly elevated (Figure 3C). However, compared with the LPS+PBS group, these inflammatory markers were markedly reduced in both the fATMSCs and DFO-fATMSCs treatment groups, with the DFO-preconditioned group demonstrating superior therapeutic efficacy over the fATMSCs group. Similar reductions were observed in the serum levels of TNF-α, as measured by ELISA (Figure 3D).

Figure 3.
Figure 3.

Establishment and analysis of an acute lung injury (ALI) mouse model after fATMSCs and DFO-fATMSCs treatment. (A) Development method of ALI mouse model (created in BioRender). (B) Comparison of sick score in ALI mouse Models among LPS+PBS, LPS+fATMSCs, and LPS+DFO-fATMSCs groups. (C) Quantitative real-time PCR analysis of gene expression levels of tumor necrosis factor-α (Tnf-α), interleukin-1β (Il1b), and interleukin-6 (Il6) in mouse lung tissue (naïve: untreated group). (D) The concentration of TNF-α in mouse serum was analyzed using ELISA. Data are expressed as the mean±standard deviation. Values with different letters are significantly different at p<0.05 according to one-way analysis of variance.

Histological analysis of lung sections revealed distinct pathological changes among the experimental groups. Key parameters, such as alveolar wall thickness, neutrophil infiltration, and lung wall hemorrhage, were assessed (Figure 4A and B). In the LPS+PBS group, the normal alveolar architecture was severely disrupted, characterized by significantly thickened alveolar septa, extensive neutrophil infiltration in the interstitial space and alveolar lumen, and marked congestion. In contrast, the LPS+fATMSCs group showed partial recovery, with visible alveolar structures, although thickened septa, neutrophil infiltration, and hemorrhages persisted. Compared to the LPS+PBS group and LPS+fATMSCs groups, the LPS+DFO-fATMSCs group demonstrated moderate improvement, with identifiable alveolar structures, mildly thickened septa, and fewer instances of neutrophil infiltration and hemorrhages.

Figure 4.
Figure 4.
Figure 4.

Histopathological analysis of a mouse model of acute lung injury. (A) Histological examination of lung tissue stained with hematoxylin and eosin at 200× and 400× magnification, demonstrating structural changes across different treatment groups: naïve (untreated group), lipopolysaccharide + phosphate-buffered saline (LPS+PBS), LPS + feline adipose tissue-derived mesenchymal stem cells (fATMSCs), and LPS + deferoxamine (DFO)-preconditioned fATMSCs. (B) Evaluation and comparison of thickness of alveolar, infiltration of neutrophil, and alveolar congestion in all groups of lung tissue. Data are expressed as the mean±standard deviation. Values with different letters are significantly different at p<0.05 according to one-way analysis of variance.

Preclinical safety assessment of DFO preconditioned fATMSCs. To assess the preclinical safety profile of DFO preconditioned fATMSCs, the cells were injected into three healthy felines, followed by comprehensive physical, hematological, and serological evaluations. As shown in Table II and Table III, no significant changes were observed in any of the parameters measured before and after the injections, indicating the absence of abnormalities attributable to the cell therapy. Vital signs, including heart rate, respiratory rate, and body temperature, remained stable with no abnormal variations (Figure 5A). Furthermore, serum amyloid A assays of blood samples showed no evidence of an inflammatory response (Figure 5B).

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Table II.

Complete blood count results before and after 1h injection of deferoxamine preconditioned canine adipose tissue-derived mesenchymal stem cells.

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Table III.

Serum chemistry results before and after injection of deferoxamine-conditioned feline adipose tissue-derived mesenchymal stem cells.

Figure 5.
Figure 5.
Figure 5.

Preclinical safety evaluation of deferoxamine (DFO)-preconditioned feline adipose tissue-derived mesenchymal stem cells (fATMSCs). (A) Assessment of vital physiological indices (namely heart rate, respiratory rate, and body temperature) following 1 h after injection of DFO-Preconditioned fATMSCs. (B) Analysis of feline serum amyloid A (fsAA) levels as an indicator of the inflammatory response 1 h after administration of DFO-preconditioned fATMSCs. (C) Ultrasonographic examination of major abdominal organs, including the kidneys, urinary bladder, liver, and spleen, 30 (30d) and 90 days (90d) post-injection. Data are expressed as the mean±standard deviation. ns: No statistically significant difference by Student’s t-test.

Follow-up evaluations were conducted at 30 and 90 days post-injection using abdominal ultrasonography. Renal ultrasounds of the three cats displayed normal features, including uniform renal echotexture, smooth contours, and a distinct corticomedullary junction, with no signs of stones or tumors. The bladder wall was of normal thickness, with a well-defined mucosal interface and no intraluminal masses. No abnormalities were detected in the bladder content or trigone region. Liver ultrasounds showed a liver of normal size, with a homogeneous echogenic pattern and no vascular irregularities or lesions. Gallbladder scans revealed a well-dilated lumen, normal wall thickness, and an absence of deposits or stones, with the common bile duct not visualized. Splenic ultrasounds confirmed normal size, intact capsule, and typical positioning, with homogeneous parenchyma and echogenicity consistent with normal criteria (Figure 5C).

Discussion

Recently, fATMSCs with their unique ability to differentiate into various tissue cells and their superior maintenance of stemness and differentiation potential compared to other stem cell types, have emerged as a key focus in regenerative medicine and tissue engineering research (23, 24). Extensive research is currently being conducted to investigate the therapeutic potential and differentiation capabilities of these cells, with a focus on enhancing their therapeutic efficacy and safety through various pretreatment strategies (25, 26). In particular, cytokine and hypoxia preconditioning have been identified as crucial methods in this context (25, 27). Consequently, this study aimed to explore the effects of DFO preconditioning on fATMSCs and assess its potential to improve their therapeutic efficacy and preclinical safety.

Prior to assessing the impact of DFO on fATMSCs, the isolated cells underwent comprehensive characterization. Flow cytometry analysis revealed that the majority of passage 3 cells expressed the well-established stem cell surface markers CD9 and CD44, while a minority expressed CD34 and CD45. Furthermore, we characterized the differentiation capacity of the cells and confirmed that fATMSCs possess multidirectional differentiation potential. Consequently, the cells utilized in this study were conclusively identified as fATMSCs (28).

To assess the effect of DFO pretreatment on the immunomodulatory potential of fATMSCs, we conducted an analysis of the expression profiles of key immunomodulatory genes using qRT-PCR. The results indicated a significant increase in the expression levels of critical immunomodulation-related genes, specifically Tsg-6 and Hgf, in DFO-pretreated cells. Hgf mitigates inflammatory responses by inhibiting cell proliferation and reducing the secretion of pro-inflammatory factors, thereby contributing to tissue repair and regeneration (29, 30). Tsg-6 plays a role in modulating immune responses by inhibiting the migration and activation of inflammatory cells and can bind to matrix molecules, such as hyaluronic acid and chondroitin sulfate, to suppress inflammation progression (31, 32). Furthermore, exosomes derived from hypoxia-preconditioned MSCs exhibit high efficacy in alleviating inflammation, highlighting the potential therapeutic importance of Tsg-6 and Hgf (33, 34). Therefore, strategies such as DFO pretreatment, which enhance MSC function and promote a more robust immunosuppressive phenotype, hold significant promise for the treatment of inflammatory-related diseases, including ALI, acute pancreatitis, and inflammatory bowel disease (13, 35-37).

To further assess the therapeutic potential of DFO-pretreated fATMSCs, we utilized a mouse model of ALI induced by LPS. The results demonstrated that DFO-pretreated fATMSCs exhibited superior therapeutic efficacy compared to untreated fATMSCs in the ALI model. This was evident by the marked decrease in gene expression of proinflammatory cytokines, specifically TNF-α, IL-1β, and IL6, in lung tissues, as well as a reduction in serum TNF-α concentration. These results indicate that DFO pretreatment created hypoxic conditions conducive to stem cell growth, and hypoxic preconditioning enhanced the immunomodulatory capacity of fATMSCs, potentially through increasing the secretion of hepatocyte growth factor (HGF) and tumor necrosis factor-stimulated gene 6 (TSG-6), thereby inhibiting the release of inflammatory cytokines (33, 34, 38, 39).

In this study, in vivo investigations have revealed that pretreating fATMSCs with DFO enhances their efficacy in mitigating the inflammatory response. Consequently, it is imperative to extend these findings to assess the clinical safety of these cells, ensuring the practicality of this therapeutic approach. The clinical safety evaluation encompassed physical examinations, hematological and serological assessments, feline serum amyloid A (fsAA) analysis, and an assessment of tumorigenic potential. Clinical data indicated that the administration of DFO-pretreated fATMSCs via injections did not elicit any adverse effects in healthy individuals. Follow-up imaging assessments performed one and three months post-injection did not detect any abnormal lesions, including tumor formation, in various tissues or organs. These findings are pivotal as they affirm the immediate and short-term safety of utilizing DFO-pretreated fATMSCs in a therapeutic setting.

The investigation into the therapeutic applications of fATMSCs has significantly progressed through the examination of the effects of DFO pretreatment on fATMSCs. This study has elucidated the augmenting impact of DFO pretreatment on the therapeutic efficacy of MSCs, particularly in reducing inflammation. It has also laid the groundwork for understanding the clinical safety aspects, underscoring the feasibility of translating this innovative therapeutic approach. While the outcomes of this study are promising, further in-depth research is necessary to delve into the clinical safety of this intervention. The therapeutic effectiveness of a single injection may be limited for stubborn diseases, and concerns persist regarding the safety of multiple administrations of the drug and potential side effects.

Above all, this study highlights the potential of DFO pretreatment as a viable strategy to enhance the efficacy of fATMSCs while upholding a favorable safety profile. The translation of laboratory findings to clinical practice is a complex and demanding process, but the positive outcomes observed in this study present an optimistic outlook for the future of regenerative medicine. As research advances, a deeper understanding of the mechanisms of action of MSC therapies will be attained, and innovative approaches like DFO preconditioning will undoubtedly introduce new concepts for future investigations, playing a pivotal role in the advancement of this field.

Conclusion

Pretreatment with DFO significantly enhanced the expression of Tgfβ, Hgf, and Tsg-6 genes in fATMSCs. In an ALI mouse model, DFO-pretreated fATMSCs showed enhanced anti-inflammatory effects, reducing inflammatory cytokines. Additionally, a single injection of these cells in felines was safe, with no immediate inflammatory responses, structural changes, or tumorigenesis observed. Hence, DFO-pretreated fATMSCs may present a safe and effective therapeutic approach for veterinary inflammatory conditions.

Footnotes

  • Authors’ Contributions

    HLT and MKC contributed to conceptualization, investigation, resources, and writing of the original draft; ZQM, XPJ and SYJ contributed to reviewing and editing; YZZ, MFX, XZL, and HSA contributed to experimental advice and reviewing; SHC and QL contributed to supervising the procedures. All authors have read and agreed to the published version of the article.

  • Conflicts of Interest

    All Authors disclosed no relevant relationships.

  • Funding

    This research was supported by “Regional Innovation Strategy (RIS)” through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2021RIS-001). This work also was supported by the Science and Technology Development Project in Jilin Province (YDZJ202201ZYTS436).

  • Received October 21, 2024.
  • Revision received November 13, 2024.
  • Accepted November 15, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Preclinical Assessment of Safety and Efficacy of Deferoxamine (DFO)-pretreated Feline Adipose Mesenchymal Stem Cells
HAILONG TENG, MINKYUNG CHO, ZHIQIANG MA, XINPENG JI, SHIYU JIN, YUZE ZHOU, MEIFU XUAN, HYUN-A SEONG, XIANGZI LI, SEONG-HO CHOI, QIANG LI
In Vivo Jan 2025, 39 (1) 267-279; DOI: 10.21873/invivo.13825
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