JAHP_14_2_428-434

Introduction

Adipose-derived stem cells (ASCs) are adult multipotent cells with strong homing abilities, immunomodulatory properties, and the capacity to promote tissue repair (Bourin et al., 2013). Their clinical application is also more ethically acceptable compared to embryonic stem cells, as their collection is minimally invasive and free from major moral concerns (Dominici et al., 2006; Yaseen et al., 2025). Adipose tissue is an excellent source of autologous mesenchymal stem cells (MSCs) and can be harvested more easily than bone marrow–derived stem cells, with fewer complications for the donor. This advantage is partly due to their lack of MHC-II expression and their low levels of MHC-I proteins, reducing the risk of immune rejection (Zuk et al., 2001).

Furthermore, several studies (Liqing et al., 2011; Bajek et al., 2017; Obaid et al., 2025; Bourin et al., 2013) have highlighted the growing interest in adipose-derived stem cells for regenerative medicine. These studies emphasize the need to optimize isolation techniques, characterization standards, culture conditions, and differentiation protocols to ensure genetic stability, reproducible therapeutic effects, and overall safety of ASC-based therapies. Bone marrow stromal cells have also been shown to differentiate into Schwann cell–like cells capable of stimulating peripheral nerve regeneration. Several studies further demonstrate that these cells can migrate to the dorsal root ganglia and spinal cord (Rodriguez et al., 2019; Obaid et al., 2020) and can form microglial and neuroectodermal cell lineages (Brayfield et al., 2010; Yaseen et al., 2024).

Therefore, the objective of this study was to examine the effect of transplanting neuron-like stem cells on the regeneration of sciatic nerve neurotmesis in rats.

Materials and Methods

Experimental design

The study was conducted with the ethical approval of the Ethics Committee of the Biotechnology Research Center, Al-Nahrain University, under decision number E.B.B.16 dated 2.1.2024. The experimental investigation included sixteen adult male rats, aged 8–12 weeks and weighing approximately ±20 g (Essa et al., 2020).

In the control group, 10 mL of PBS buffer was administered perineurally to treat the defect. In the MSC-treated group, rats received a perineural transplantation of 5 × 10⁶ cells. All animals were housed in the central animal care facility and provided with free access to food and water throughout the study period. On the 112th postoperative day, all animals from both groups were euthanized for histological evaluation of sciatic nerve regeneration.

In vitro study

Isolation and cultivation of AD-MSCs

AD-MSCs were isolated following the method described by Zedan and Al-Ameri (2023). The cells were prepared in the Tissue Culture Unit, Department of Biotechnology, Al-Nahrain University. Briefly, under anesthesia, subcutaneous adipose tissue was aseptically excised from the experimental rat (Figure 1). The tissue was thoroughly washed in phosphate-buffered saline (PBS) and then minced into small fragments. It was digested with collagenase type I (1 mg/mL; Gibco, USA) for 1 hour at 37°C.

After digestion, the suspension was filtered and cultured. The medium was replaced after 72 hours, and non-adherent cells were discarded. When the culture reached confluence on day 12, the monolayer was rinsed twice with 2 mL of PBS (pH 7.2). A solution of 0.20% trypsin–0.02% EDTA (Sigma, USA) was added and incubated for 2 minutes to detach the cells. DMEM supplemented with 10% FBS was added to neutralize trypsin, and the detached cells were gently aspirated. The cell suspension was transferred to sterile tubes and centrifuged at 2000 rpm for 5 minutes.

The resulting pellet was resuspended in 1 mL of DMEM. The cell count was determined using a hemocytometer to obtain a final concentration of 5 × 10⁶ AD-MSCs.

Immunohistochemistry analysis of MSCs

After MSCs were suspended in DMEM growth medium and dissociated using trypsin–PBS, the cells were re-cultured in multi-well tissue culture slides. Eight wells were seeded in DMEM supplemented with 15% FBS, and the plates were incubated at 37 °C to allow the cells to form a monolayer within 1–3 days. The medium was then removed, and the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature (20–25 °C) in a humid chamber.

Following fixation, the cells were washed with PBS and incubated with 1% hydrogen peroxide for 10–15 minutes to block endogenous peroxidase activity. To minimize background staining, 1.5% blocking serum was applied for 1 hour. The cells were then rinsed with PBS (three times, 5 minutes each).

Next, the samples were incubated with 125 µL of diluted primary antibody and then washed again three times with PBS. This was followed by incubation with 1.2 mL of biotinylated secondary antibody for 30 minutes. After washing, 650 µL of ABC reagent was added to the wells, followed by additional PBS rinsing.

For visualization, the cells were treated with peroxidase substrate (three drops) for approximately 10 minutes, or until adequate stain intensity was achieved, and then rinsed with purified water. The samples were counterstained with hematoxylin for 5–10 seconds and washed again.

Finally, one to two drops of permanent mounting medium were applied, and the slides were examined under a light microscope at 40× and 100× magnification (Bucan et al., 2019).

Neurogenic differentiation

Neuronal induction of AD-MSCs was performed using a modified protocol based on published methods (Ying et al., 2012). After 24 hours of incubation, the culture medium was removed and replaced with serum-free MEM containing 2 mM β-mercaptoethanol (BME) for 1 hour. The medium was then discarded, and the cells were washed with serum-free MEM.

For the induction phase, serum-free MEM supplemented with 1 mM retinoic acid (Gibco, USA), 10 ng/mL nerve growth factor (NGF) (Gibco, USA), and 0.1 ng/mL sonic hedgehog (Shh) (Gibco, USA) was added. Cells were incubated at 37°C for four days under these conditions.

As a negative control, a parallel set of MSCs was cultured under the same environmental conditions but without induction factors. All cultures were maintained in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C, and the medium was replaced every two days.

Assessment of neurogenic differentiation

Histological examination was performed after 21 days. The pellets were fixed in 4% buffered formalin at 4 °C for short-term fixation, then dehydrated in ethanol and embedded in paraffin. Sections with a thickness of 4 µm were cut from the paraffin blocks, deparaffinized with xylene, and rehydrated with tap water. The obtained sections were stained with hematoxylin and eosin (H&E) to visualize cell morphology.

In vivo animal study

Surgical procedure of sciatic nerve injury

Each mouse was anesthetized with a combination of drugs at a dose of 75 mg/kg. The left rump was prepared aseptically. A longitudinal incision was made on the posterolateral thigh, approximately 1 cm caudolateral to the greater trochanter, exposing about 33% of the distal femur. The subcutaneous tissue and fascia lata were carefully dissected. Through this skin entry point, the cranial portion of the biceps femoris and the posterior portion of the semitendinosus muscles were separated using blunt dissection with Mayo scissors to expose the sciatic nerve and isolate it from the surrounding tissues.

As previously described, a hemostatic forceps was applied for 60 seconds to induce an axonotmesis (crush) injury (Liu et al., 2020). Using a Hamilton microsyringe, 106 cells in suspension were transplanted perineurally into the MSC site. After transplantation, the musculature and skin were sutured. The mouse was then allowed to recover in a clean enclosure on a 37 °C warming pad. Antimicrobials (gentamicin, 100 mg/kg, i.m.) were administered daily for 7 days post-injury.

Histological investigation of sciatic nerve repair

The samples underwent standard histological procedures on the 12th day after surgery. Tissue sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope for histological alterations (Luna, 1992).

Results

Morphology of bone marrow-derived mesenchymal stem cells

Small cell colonies were observed in the experimental culture 48–72 hours after seeding. The cells were round and flattened, and they began to attach to the plastic surface of the culture flask in their initial state (Figure 2A). After ten days, a monolayer of adherent cells was formed, during which the colonies and individual cells expanded, and the cells gradually increased in size and interconnected. After 12 days of growth at passage 1 (P1), the cells exhibited a large, flattened, spindle-shaped, and slightly irregular morphology (Figure 2B). Two days after subculture at passage 2 (P2), the AD-MSCs showed a high proportion of spindle-shaped cells that aligned along their longitudinal axis. By five days post-subculture at passage 3 (P3), the AD-MSCs displayed a heterogeneous morphology, including large, flattened, spindle-shaped, round, and polygonal cells (Figure 2C).

Immunophenotypic analysis of MSCs

Immunocytochemical staining was used to rapidly identify MSCs from a mixed cell suspension. This analysis revealed that most cells expressed the characteristic MSC markers (Figure 3A). In contrast, the cells were negative for CD34 and were distinctly counterstained blue with Harris hematoxylin (Figure 3B).

Histological analysis of MSCs

Changes in post-seeding morphology are shown in Figure 4. By the eighth day after seeding, the cells became smaller and more compact, forming bipolar or multipolar processes extending from the cell body. In some cells, a single long axon-like projection was observed, and certain neurons exhibited a pyramidal shape. Neural morphology was distinguished from typical cell morphology based on the refractive appearance of the cells, the presence of bipolar or multipolar neurite-like projections (with at least one projection equal to or longer than the soma), and the occasional presence of cellular extensions.

Histological evaluation of nerve regeneration

In the control group, the proximal stump showed relatively well-aligned regenerative nerve fibers, mild adhesion to surrounding tissue, vacuolated nerve fibers, collagen deposition (scar formation) in the perineurium and epineurium, and evidence of remyelination of regenerative nerve fibers (Figure 5A). Histological examination of the middle stump revealed a reduced number of nerve fibers, mild proliferation of Schwann cells, improved parallel alignment of nerve fibers, and the presence of fibrous tissue and inflammatory cells (Figure 5B). The distal stump displayed vacuolated, degenerated nerve fibers, minimal scar tissue, improved myelination, and inflammatory cell infiltration (Figure 5C).

In the MSC-treated group, the proximal stump demonstrated enhanced parallel alignment of nerve fibers, minimal residual endoneurial channels, robust remyelination of regenerative nerve fibers, and active Schwann cells (Figure 5D). The middle stump exhibited active Schwann cells, parallel arrangement of nerve fibers, formation of new blood vessels, remyelination of regenerative fibers, and few inflammatory cells (Figure 5E). In the distal stump, vacuolated degenerating nerve fibers were observed along with well-aligned regenerative nerve fibers and active Schwann cells (Figure 5F).

Discussion

Transplantation of neural stem cells (NSCs) has been proposed as a potential therapeutic approach for neurodegenerative disorders (Phinney and Prockop, 2007). However, the need for immunosuppressive regimens in allogeneic transplantation and the limited availability of donor brain tissue pose significant challenges. These limitations could be overcome if NSCs were derived from clinically accessible autologous sources, such as adipose tissue or peripheral blood. Recently, mesenchymal stem cells (MSCs) have been shown to promote angiogenesis, neurogenesis, and the repair of injured tissues (Jang et al., 2016; Obaid et al., 2020; Kareem and Hussein, 2023).

In the present study, MSCs were characterized by their morphology and confirmed by surface marker analysis, showing 98.9% positivity for CD90. This is consistent with previous reports (Fu et al., 2008) demonstrating that MSCs express characteristic antigens including CD29, CD44, CD73, CD90, CD105, and CD166, while lacking hematopoietic markers such as CD34, CD45, CD14, or CD11. Similarly, Marconi et al. (2012), Li et al. (2017), and Khoo et al. (2011) reported positive CD90 and negative CD34 expression in MSCs, consistent with our findings regarding homing receptor-mediated migration of hematopoietic and bone marrow stem cells.

For neural differentiation, a combination of growth factors and retinoic acid (RA) was employed. Growth factor-based differentiation has shown promising results in previous studies (Maden, 2007). However, chemical-based methods remain controversial due to the potential cytotoxic effects of these compounds on cell size and morphology (Montiel-Eulefi et al., 2012). RA, an active metabolite of vitamin A, is widely expressed in the nervous system during development and acts as a potent inducer of differentiation, with receptors and binding proteins throughout the brain, suggesting a critical role in neural development and function. These findings align with Varejão et al. (2001), who successfully used RA and other neural-inducing agents to generate neural-like cells.

Histomorphological analysis in this study revealed that transplantation of AD-MSCs enhanced the formation of nerve fibers and angiogenesis, suggesting a therapeutic role in nerve repair. These results are consistent with Altemeemi et al. (2021), who demonstrated that MSC implantation, either alone or in combination with guiding conduits, can improve peripheral nerve regeneration in cases of neurotmesis. Similarly, Cartarozzi et al. (2015) and Hameed et al. (2011) reported that stem cell therapy promotes neovascularization through multiple mechanisms, including differentiation of transplanted cells into endothelial cells and secretion of trophic or angiogenic factors (Zhang et al., 2017; Al-Maliki et al., 2025). Vascular endothelial growth factor (VEGF) is particularly important, influencing astrocytes, Schwann cells, microglia, and neural progenitors. VEGF can protect motor neurons from oxidative stress and hypoxia-induced apoptosis (Sarikcioglu et al., 2009; Zedan and Al-Ameri, 2022), stimulate axonal growth, and enhance the survival of neurons and satellite cells in dorsal root ganglia (Zachary, 2005).

Histological observations in the MSC-treated group showed minimal intraneural scarring, whereas the control group exhibited extensive collagen deposition. This suggests that axonal regeneration is influenced by a balance between regeneration and scar formation. Supporting this, studies by Varejão et al. (2004) and Sondell et al. (1999) demonstrated that interventions reducing scar formation, such as tissue plasminogen activator (tPA), enhance axonal regeneration and myelination in the peripheral nervous system. Collectively, these findings emphasize the potential of MSC therapy to promote neural repair while minimizing inhibitory effects of scar tissue.

Conclusions

Transplantation of neural-differentiated adipose-derived mesenchymal stromal cells significantly enhances axonal regeneration and limits scar formation following sciatic nerve crush injury in mice, highlighting their promising therapeutic potential for peripheral nerve repair.

Acknowledgement

Thanks only to the authors for providing their time, effort and support for the manuscript.

NOVELTY STATEMENT

This study is the first to demonstrate that adipose-derived mesenchymal stromal cells (AD-MSCs), pre-stimulated for neural differentiation and angiogenesis, can effectively enhance sciatic nerve regeneration.

AUTHOR’S CONTRIBUTION

All authors contributed equally to the manuscript, including study design, manuscript writing, linguistic editing, and journal selection.

Funding sources

This study received no funding from any institution, university, or organization; all support was provided solely by the authors.

Generative AI and AI-assisted technology statement

No artificial intelligence tools were used in the manuscript.

Conflict of interest

The authors have declared no conflict of interest.

References

Al-Maliki NS, Jumaah YH, Hameed HM, Khudhair OE, Hadid MA, Hasan AF (2025). Evaluation of miRNA-155 as a biomarker for cancer stem cells and its role in chemotherapy resistance in Iraqi patients with acute myeloid leukemia. Opera Med. Physiol., 12(1): 30-37.

Altemeemi AS, KadhimNK, Nsaif GS (2021). Changes in interleukins and follicle stimulating hormone in toxoplasmosis male patients. Indian J. Forensic Med. Toxicol., 15(1): 803-807.

Bajek A, Gurtowska N, Olkowska, Drewa T (2017). Does the harvesting technique affect the properties of adipose-derived stem cells? The comparative biological characterization. J. Cell Biochem., 118: 1097-1107. https://doi.org/10.1002/jcb.25724

Bourin P, Bunnell BA, Casteilla L, Dominici M, Gimble JM (2013). Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: A joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy, 15: 641-648. https://doi.org/10.1016/j.jcyt.2013.02.006

Brayfield C, Marra K, Rubin JP (2010). Adipose stem cells for soft tissue regeneration. Handchirurgie Mikrochirurgie Plastische Chirurgie, 42: 124-128. https://doi.org/10.1055/s-0030-1248269

Bucan V, Vaslaitis D, Peck CT, Vogt PM, Radtke C (2019). Effect of exosomes from rat adipose-derived mesenchymal stem cells on neurite outgrowth and sciatic nerve regeneration after crush injury. Mol. Neurobiol., 56(3): 1812-1824. https://doi.org/10.1007/s12035-018-1172-z

Cartarozzi LP, Spejo AB, Ferreira Jr RS, Barraviera B, Oliveira AL (2015). Mesenchymal stem cells engrafted in a fibrin scaffold stimulates Schwann cell reactivity and axonal regeneration following sciatic nerve tubulization. J. Brain Res. Bull., 112: 14-24. https://doi.org/10.1016/j.brainresbull.2015.01.005

Dominici ML, Le Blanc K, Mueller I, Horwitz EM (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. Int. Soc. Cell. Ther. Posit. Statement Cytotherapy, 8: 315-317. https://doi.org/10.1080/14653240600855905

Essa HH, Jasim, HS, Kadhim HA (2020). Immunological and hematological response to local transplantation of stem cells in injured radial nerve of dogs. Iraqi J. Vet. Med., 44(2): 45–55. https://doi.org/10.30539/ijvm.v44i2.976

Fu L, Zhu L, Huang Y, Lee TD, Forman SJ, Shih CC (2008). Derivation of neural stem cells from mesenchymal stem cells: evidence for a bipotential stem cell population. Stem Cells Dev., 17: 1109-1122. https://doi.org/10.1089/scd.2008.0068

Hameed AT, Ibrahim R, Karim AJ, Abz Z, Ti A (2011). Clinical and histopathological observations of autologous bone marrow stromal cells implantation on the regeneration of sciatic nerve neurotmesis in rabbit. Afr. J. Biotechnol., 10(33): 6310-6318.

Jang S, Cho HH, Kim SH, Lee KH, Jeong HS (2016). Transplantation of human adipose tissue-derived stem cells for repair of injured spiral ganglion neurons in deaf guinea pigs. Neural Regen. Res., 11: 994. https://doi.org/10.4103/1673-5374.184503

Kareem KN, Hussein ZA (2023). The GGC medium reduces the DNA fragmentation of human spermatozoa via in vitro activation. Arch. Razi Inst., 78(2): 709-714.

Khoo ML, Tao H, Ma DD (2011). Transplantation of neuronal-primed human bone marrow mesenchymal stem cells in hemiparkinsonian rodents. PLoS One, 6: e19025. https://doi.org/10.1371/journal.pone.0019025

Li Y, Xu W, Cheng LY (2017). Adipose-derived mesenchymal stem cells accelerate nerve regeneration and functional recovery in a rat model of recurrent laryngeal nerve injury. Neural Regen. Res., 12: 1544. https://doi.org/10.4103/1673-5374.215267

Liqing Y, Jia G, Jiqing C, Cheng Z (2011). Directed differentiation of motor neuron cell-like cells from human adipose-derived stem cells in vitro. Neuroreport, 22: 370-373. https://doi.org/10.1097/WNR.0b013e3283469615

Liu CY, Yi, G, Sun YD, Lin YF, Xie Z, Lin HD (2020). Effect of exosomes from adipose-derived stem cells on the apoptosis of Schwann cells in peripheral nerve injury. CNS Neurosci. Therap., 26(2): 189-196. https://doi.org/10.1111/cns.13187

Luna LG (1992). Histopathologic methods and color atlas of special stains and tissue artifacts. Maryland: American Histolabs, Inc.,

Obaid MR, Tareq YF, Kareem KN, Hameed SD, Tarq SZ, Sahib AD, Hasan AF (2025). Changes in the level of lipid profile in diabetes mellitus in samples patients. J. Biosci. Appl. Res., 11(1): 331-336. https://doi.org/10.21608/jbaar.2025.350090.1134

Maden M (2007). Retinoic acid in the development regeneration and maintenance of the nervous system. Nat. Rev. Neurosci., 8: 755-765. https://doi.org/10.1038/nrn2212

Marconi S, Castiglione G, Turano E, Bonett B (2012). Human adipose-derived mesenchymal stem cells systemically injected promotes peripheral nerve regeneration in the mouse model of sciatic crush. Tissue Eng., 18: 1264-1272. https://doi.org/10.1089/ten.tea.2011.0491

Montiel-Eulefi E, Nery AA, Rodrigues LC, Sánchez R, Ulrich H (2012). Neural differentiation of rat aorta pericyte cells. Cytometry Part A, 81: 65-71. https://doi.org/10.1002/cyto.a.21152

Obaid RM, Yaseen FT, Mukhlif MY (2020). Blood cells depletion after chemotherapy in Iraqi women with breast cancer. Indian J. Forensic Med. Toxicol., 14(4): 3379-3382.

Obaid RM, Yaseen FT, Salim AK (2020). Correlation between vitamin D3 (cholecalciferol) and thyroid diseases in Iraqi patients. Ann. Trop. Med. Publ. Hlth., 23: 231-603. https://doi.org/10.36295/ASRO.2020.231603

Phinney DG, Prockop DJ (2007). Concise review: Mesenchymal stem/multipotent stromal cells: the state of Tran’s differentiation and modes of tissue repair current views. Stem Cells, 25: 2896-2902. https://doi.org/10.1634/stemcells.2007-0637

Rodriguez SDN, de Lima RLA, Amorim RM (2019). Canine adipose-derived mesenchymal stromal cells enhance neuroregeneration in a rat model of sciatic nerve crush injury. Cell Transpl., 28: 47-54. https://doi.org/10.1177/0963689718809045

Sarikcioglu L, Demirel M, Utuk A (2009). Walking track analysis: An assessment method for functional recovery after sciatic nerve injury in the rat. Folia Morphol., 68: 1-7.

Sondell M, Lundborg G, Kanje M (1999). Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and Schwann cell proliferation in the peripheral nervous system. J. Neurosci., 19: 5731-5740. https://doi.org/10.1523/JNEUROSCI.19-14-05731.1999

Varejao AS, Cabrita AM, Meek MF, Giacobini-Robecchi MG (2004). Functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp. J. Neurotrauma, 21: 1652-1670. https://doi.org/10.1089/neu.2004.21.1652

Varejao AS, Meek MF, Ferreira AJ, Patrı́cio JA, Cabrita AM (2001). Functional evaluation of peripheral nerve regeneration in the rat: Walking track analysis. J. Neurosci. Methods, 108: 1-9. https://doi.org/10.1016/S0165-0270(01)00378-8

Yaseen TF, Al-Jumaily KM. Rakad (2025). The impact of interleukin-21 and 23 serum level and gene expression in celiac disease among sample of Iraqi patients. Asian J. Dairy Food Res., 44(2): 234-239. https://doi.org/10.18805/ajdfr.DRF-476

Yaseen FT, Al-Jumaily RMK (2024). Evaluation of global DNA methylation, homocysteine and vitamin B12 levels among patients with celiac disease. Gastroenterology, 58(4): 258-263. https://doi.org/10.22141/2308-2097.58.4.2024.637

Ying C, Hu W, Cheng B, Zheng X, Li S (2012). Neural differentiation of rat adipose-derived stem cells in vitro. Cell Mol. Neurobiol., 32: 1255-1263. https://doi.org/10.1007/s10571-012-9850-2

Zachary I (2005). Neuroprotective role of vascular endothelial growth factor: Signalling mechanisms, biological function, and therapeutic potential. Neurosignals, 14: 207-221. https://doi.org/10.1159/000088637

Zedan ZK, Al-Ameri SH (2022). In vivo study of impact transplantation hematopoietic progenitor cells on induced cutaneous wound healing in rabbits model. https://doi.org/10.33899/ijvs.2021.130949.1899

Zedan ZK, Al-Ameri SHA (2023). In vitro study of primary isolation and culture of adipose-derived stem cells and induction of chondrogenic differentiation. Arch. Razi Inst., 78(1): 125.

Zhang Q, Nguyen P, Xu Q, Park W, Lee S, Furuhashi A (2017). Neural progenitor like cells induced from human gingiva‐derived mesenchymal stem cells regulate myelination of Schwann cells in rat sciatic nerve regeneration. Stem Cells Transl. Med., 6: 458-470. https://doi.org/10.5966/sctm.2016-0177

Zuk PA, Zhu MN, Mizuno H, Huang J, Futrell JW, Hedrick MH (2001). Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng., 7: 211-228. https://doi.org/10.1089/107632701300062859