Interplay between mesenchymal stromal cells and the immune system after transplantation: implications for advanced cell therapy in the retina

Advanced mesenchymal stromal cell-based therapies for neurodegenerative diseases are widely investigated in preclinical models.Mesenchymal stromal cells are well positioned as therapeutics because they address the underlying mechanisms of neurodegeneration,namely trophic factor deprivation and neuroinflammation.Most studies have focused on the beneficial effects of mesenchymal stromal cell transplantation on neuronal survival or functional improvement.However,little attention has been paid to the interaction between mesenchymal stromal cells and the host immune system due to the immunomodulatory properties of mesenchymal stromal cells and the long-held belief of the immunoprivileged status of the central nervous system.Here,we review the crosstalk between mesenchymal stromal cells and the immune system in general and in the context of the central nervous system,focusing on recent work in the retina and the importance of the type of transplantation.

O-linkedβ-N-acetylglucosaminylation may be a key regulatory factor in promoting osteogenic differentiation of bone marrow mesenchymal stromal cells

Cumulative evidence suggests that O-linkedβ-N-acetylglucosaminylation(OGlcNAcylation)plays an important regulatory role in pathophysiological processes.Although the regulatory mechanisms of O-GlcNAcylation in tumors have been gradually elucidated,the potential mechanisms of O-GlcNAcylation in bone metabolism,particularly,in the osteogenic differentiation of bone marrow mesenchymal stromal cells(BMSCs)remains unexplored.In this study,the literature related to O-GlcNAcylation and BMSC osteogenic differentiation was reviewed,assuming that it could trigger more scholars to focus on research related to OGlcNAcylation and bone metabolism and provide insights into the development of novel therapeutic targets for bone metabolism disorders such as osteoporosis.

Exosomes from umbilical cord mesenchymal stromal cells promote the collagen production of fibroblasts from pelvic organ prolapse

BACKGROUND Pelvic organ prolapse(POP)involves pelvic organ herniation into the vagina due to pelvic floor tissue laxity,and vaginal structure is an essential factor.In POP,the vaginal walls exhibit abnormal collagen distribution and decreased fibroblast levels and functions.The intricate etiology of POP and the prohibition of trans-vaginal meshes in pelvic reconstruction surgery present challenges in targeted therapy development.Human umbilical cord mesenchymal stromal cells(hucMSCs)present limitations,but their exosomes(hucMSC-Exo)are promising therapeutic tools for promoting fibroblast proliferation and extracellular matrix remodeling.suppressed inflammation in POP group fibroblasts,stimulated primary fibroblast growth,and elevated collagen I(Col1)production in vitro.High-throughput RNA-seq of fibroblasts treated with hucMSC-Exo and miRNA sequencing of hucMSC-Exo revealed that abundant exosomal miRNAs downregulated matrix metalloproteinase 11(MMP11)expression.CONCLUSION HucMSC-Exo normalized the growth and function of primary fibroblasts from patients with POP by promoting cell growth and Col1 expression in vitro.Abundant miRNAs in hucMSC-Exo targeted and downregulated MMP11 expression.HucMSC-Exo-based therapy may be ideal for safely and effectively treating POP.

Mesenchymal stromal cells modulate unfolded protein response and preserve β-cell mass in type 1 diabetes

Introduction:Transplantation of mesenchymal stromal cells(MSCs)is a promising therapy for type 1 diabetes(T1D).However,whether the infused MSCs affect the endoplasmic reticulum stress or subsequent unfolded protein response inβcells remains unclear.Methods:To investigate this,we induced early-onset T1D in non-obese diabetic mice using streptozotocin.Subsequently,T1D mice were randomly assigned to receive either MSCs or phosphate-buffered saline.We observed the in vivo homing of MSCs and assessed their effectiveness by analyzing blood glucose levels,body weight,histopathology,pancreatic protein expression,and serum levels of cytokines,proinsulin,and C-peptide.Results:Infused MSCs were found in the lungs,liver,spleen,and pancreas of T1D mice.They exhibited various effects,including reducing blood glucose levels,regulating immunity,inhibiting inflammation,increasingβ-cell areas,and reducing the expression of key proteins in the unfolded protein response pathway.Fasting serum proinsulin and C-peptide levels were significantly higher in the MSCs treatment group than in the T1D model group.However,there was no significant difference in the biomarker ofβ-cell endoplasmic reticulum stress,the ratio of fasting serum proinsulin to C-peptide,between the two groups.Conclusion:Ourfindings reveal that MSCs infusion does not alleviate endoplasmic reticulum stress inβcells directly but modulates the unfolded protein response pathway to preserveβ-cell mass and function in T1D mice.

Human dental pulp stem/stromal cells in clinical practice

Dental pulp stem/stromal cells(DPSCs)are fibroblast-like,neural crest-derived,and multipotent cells that can differentiate into several lineages.They are relatively easy to isolate from healthy and inflamed pulps,with little ethical concerns and can be successfully cryopreserved and thawed.The therapeutic effects of DPSCs derived from animal or human sources have been extensively studied through in-vitro and in-vivo animal experiments and the findings indicated that DPSCs are effective not only for dental diseases but also for systemic diseases.Understanding that translational research is a critical step through which the fundamental scientific discoveries could be translated into applicable diagnostics and therapeutics that directly benefit humans,several clinical studies were carried out to generate evidence for the efficacy and safety of autogenous or allogeneic human DPSCs(hDPSCs)as a treatment modality for use in cell-based therapy,regenerative medicine/dentistry and tissue engineering.In clinical medicine,hDPSCs were effective for treating acute ischemic stroke and human exfoliated deciduous teeth-conditioned medium(SHED-CM)repaired vascular damage of the corpus cavernous,which is the main cause of erectile dysfunction.Whereas in clinical dentistry,autologous SHED was able to rege-nerate necrotic dental pulp after implantation into injured teeth,and micrografts enriched with autologous hDPSCs and collagen sponge were considered a treatment option for human intrabony defects.In contrast,hDPSCs did not add a significant regenerative effect when they were used for the treatment of post-extraction sockets.Large-scale clinical studies across diverse populations are still lacking to provide robust evidence on the safety and efficacy of hDPSCs as a new treatment option for various human diseases including dental-related problems.

Non-enzymatic methods for isolation of stromal vascular fraction and adipose-derived stem cells:A systematic review

BACKGROUND Adipose-derived stem cells(ADSCs)and the stromal vascular fraction(SVF)have garnered substantial interest in regenerative medicine due to their potential to treat a wide range of conditions.Traditional enzymatic methods for isolating these cells face challenges such as high costs,lengthy processing time,and regulatory complexities.AIM This systematic review aimed to assess the efficacy and practicality of nonenzymatic,mechanical methods for isolating SVF and ADSCs,comparing these to conventional enzymatic approaches.METHODS Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines,a comprehensive literature search was conducted across multiple databases.Studies were selected based on inclusion criteria focused on non-enzymatic isolation methods for SVF and ADSCs from adipose tissue.The risk of bias was assessed,and a qualitative synthesis of findings was performed due to the methodological heterogeneity of the included studies.RESULTS Nineteen studies met the inclusion criteria,highlighting various mechanical techniques such as centrifugation,vortexing,and ultrasonic cavitation.The review identified significant variability in cell yield and viability,and the integrity of isolated cells across different non-enzymatic methods compared to enzymatic procedures.Despite some advantages of mechanical methods,including reduced processing time and avoidance of enzymatic reagents,the evidence suggests a need for optimization to match the cell quality and therapeutic efficacy achievable with enzymatic isolation.CONCLUSION Non-enzymatic,mechanical methods offer a promising alternative to enzymatic isolation of SVF and ADSCs,potentially simplifying the isolation process and reducing regulatory hurdles.However,further research is necessary to standardize these techniques and ensure consistent,high-quality cell yields for clinical applications.The development of efficient,safe,and reproducible non-enzymatic isolation methods could significantly advance the field of regenerative medicine.

Mesenchymal Stromal Cells and Their Uses in Bio-Regenerative Therapies for Bone and Cartilage: A Review

Mesenchymal stromal cells (MSCs) are a top candidate for new clinical treatments in the repair of bone and cartilage. In several clinical trials, they have shown reliable, effective, and safe management of inflammation, pain, and the regenerative capabilities of resident tissues. MSCs are likely derived from pericytes. They modulate the environment they are placed in by secreting immunomodulatory and signaling molecules to reduce inflammation and direct resident cells to create new tissues. They are easily isolated from several different adult tissues, and inexpensive to grow in a lab. However, a mistake made in the initial classification of MSCs as stem cells has created deeply engrained misconceptions that are still evident today. MSCs are not stem cells, despite a large fraction of research and therapies using the name “mesenchymal stem cells”. This mistake creates false narratives attributing the observed positive outcomes of MSC treatments to stem cell characteristics, which has led to distrust in MSC research. Despite inconsistencies in their classification, MSCs demonstrate consistent positive effects in numerous animal studies and human clinical trials for non-unions and osteoarthritis. With an aging population, regenerative techniques are very promising for novel therapies. To produce trusted and safe new treatments using MSCs, it is essential for the International Society for Cellular Therapies to re-establish common ground in the identity, mechanism of action, and isolation techniques of these cells.

Isolation, Cuture and Biological Characteristic Analysis of Stromal and Glandular Epithelial Cells of Buffalo (Bubalus bubalis) Endometrium

[Objective] The experiment aimed to set up a method for isolating and culturing endometrial stromal cells (BESC) and endometrial glandular epithelial cells(BEGEC) of buffalo as well as laid foundation for studying biological mechanism of embryo implantation and uterine diseases. [Method] The enzymatic digestion method, scraping method, serial filtration and differential velocity adherent technique were used to isolate BESC and BEGEC, then immunocytochemical method and TRYPAN-Blue assay were used to determine the purity and survival rate of isolated cells. [Result] The BESC and BEGEC were successfully isolated and cultured while immunocytochemical method and cell count method demonstrated that the purity was over 90%. The result of TRYPAN-Blue assay shown that survival rate of BESC and BEGEC was 91% and 78% respectively. [Conclusion] The enzymatic digestion method, scraping method, serial filtration and differential velocity adherent technique could isolate BESC and BEGEC with high purity.

Overexpression of lentivirus-mediated glial cell line-derived neurotrophic factor in bone marrow stromal cells and its neuroprotection for the PC12 cells damaged by lactacystin

Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor （GDNF）, and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells （BMSCs）. Methods pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. Results Virus stock of GDNF was harvested with the titer of 5.6×10^5 TU/mL. After tmnsduction, GDNF-BMSCs successfully secreted GDNF to supematant with nigher concentration （800 pg/mL） than BMSCs did （less than 100 pg/mL）. The supematant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin （10 μmol/L）. Conclusion Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.

Bone Marrow Stromal Cells Express Neural Phenotypes in vitro and Migrate in Brain After Transplantation in vivo

Objective To investigate the differentiation of bone marrow stromal cells （BMSC） into neuron-like cells and to explore their potential use for neural transplantation. Methods BMSC from rats and adult humans were cultured in serum-containing media. Salvia miltiorrhiza was used to induce human BMSC （hBMSC） to differentiate. BMSC were identified with immunocytochemistry. Semi-quantitative RT-PCR was used to examine mRNA expression of neurofilamentl （NF1）, nestin and neuron-specific enolase （NSE） in rat BMSC （rBMSC）. Rat BMSC labelled by Hoschst33258 were transplanted into striatum of rats to trace migration and distribution. Results rBMSC expressed NSE, NFI and nestin mRNA, and NF1 mRNA and expression was increased with induction of Salvia miltiorrhiza. A small number of hBMSC were stained by anti-nestin, anti-GFAP and anti-S 100. Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. Some differentiated neuron-like cells, that expressed NSE, beta-tubulin and NF-200, showed typical neuron morphology, but some neuron-like cells also expressed alpha smooth muscle protein, making their neuron identification complicated, rBMSC could migrate and adapted in the host brains after being transplanted. Conclusion Bone marrow stromal cells could express phenotypes of neurons, and Salvia milliorrhiza could induce hBMSC to differentiate into neuron-like cells, If BMSC could be converted into neurons instead of mesenchymal derivatives, they would be an abundant and accessible cellular source to treat a variety of neurological diseases.

Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy?

Mesenchymal stromal cells(MSCs) are currently being investigated for use in a wide variety of clinical applications. For most of these applications, systemic delivery of the cells is preferred. However, this requires the homing and migration of MSCs to a target tissue. Although MSC hominghas been described, this process does not appear to be highly efficacious because only a few cells reach the target tissue and remain there after systemic administration. This has been ascribed to low expression levels of homing molecules, the loss of expression of such molecules during expansion, and the heterogeneity of MSCs in cultures and MSC culture protocols. To overcome these limitations, different methods to improve the homing capacity of MSCs have been examined. Here, we review the current understanding of MSC homing, with a particular focus on homing to bone marrow. In addition, we summarize the strategies that have been developed to improve this process. A better understanding of MSC biology, MSC migration and homing mechanisms will allow us to prepare MSCs with optimal homing capacities. The efficacy of therapeutic applications is dependent on efficient delivery of the cells and can, therefore, only benefit from better insights into the homing mechanisms.

Ultrastructure of neuronal-like cells differentiated from adult adipose-derived stromal cells

β-mercaptoethanol induces in vitro adult adipose-derived stromal cells （ADSCs） to differentiate into neurons. However, the ultrastructural features of the differentiated neuronal-like cells remain unknown. In the present study, inverted phase contrast microscopy was utilized to observe β-mercaptoethanol-induced differentiation of neuronal-like cells from human ADSCs, and immunocytochemistry and real-time polymerase chain reaction were employed to detect expression of a neural stem cells marker （nestin）, a neuronal marker （neuron-specific enolase）, and a glial marker （glial fibrillary acidic protein）. In addition, ultrastructure of neuronal-like cells was observed by transmission election microscopy. Results revealed highest expression rate of nestin and neuron-specific enolase at 3 and 5 hours following induced differentiation; cells in the 5-hour induction group exhibited a neuronal-specific structure, i.e., Nissl bodies. However, when induction solution was replaced by complete culture medium after 8-hour induction, the differentiated cells reverted to the fibroblast-like morphology from day 1. These results demonstrate that β-mercaptoethanol-induced ADSCs induced differentiation into neural stem cells, followed by morphology of neuronal-like cells. However, this differentiation state was not stable.

In vitro differentiation of adipose-derived stem cells and bone marrow-derived stromal stem cells into neuronal-like cells

Adipose-derived stem cells and bone marrow-derived stromal stem cells were co-cultured with untreated or Aβ1-40-treated PC12 cells, or grown in supernatant derived from untreated or Aβ1-40-treated PC12 cells. Analysis by western blot and quantitative real-time PCR showed that protein levels of Nanog, Oct4, and Sox2, and mRNA levels of miR/125a/3p were decreased, while expression of insulin-like growth factor-2 and neuron specific enolase was increased. In comparison the generation of neuron specific enolase-positive cells was most successful when adipose-derived stem cells were co-cultured with Aβ1-40-treated PC12 cells. Our results demonstrate that adipose-derived stem cells and bone marrow-derived stromal stem cells exhibit trends of neuronal-like cell differentiation after co-culture with Aβ1-40-treated PC12 cells. This process may relate to a downregulation of miR-125a-3p mRNA expression and increased levels of insulin-like growth factor-2 expression.

Over-expression of VEGF in Marrow Stromal Cells Promotes Angiogenesis in Rats with Cerebral Infarction via the Synergistic Effects of VEGF and Ang-2

This study explored whether the transplantation of modified marrow stromal cells (MSCs) has angiogenic effects in a left middle cerebral artery occlusion infarction/reperfusion (MCAO I/R) rat model and preliminarily examined the mechanism of angiogenesis following cerebral infarction.MSCs were isolated by using a direct adherent method and cultured.Vascular endothelial growth factor (VEGF) was transfected into MSCs by employing the liposome transfection.The transfection efficiency was measured by the optical density method.The protein expression of VEGF gene before and after transfection was measured by Western blotting.SD rat model of transient occlusion of the left middle cerebral artery was established by using an approach of intra-luminal occlusion.Tetrazolium (TTC) and HE staining were performed to observe the cerebral infarction.ELISAs were used to measure the levels of VEGF in the rat cerebral tissues.The expression patterns of angiopoietin-2 (Ang-2) and CD34 in cells surrounding the area of infarction were immunohistochemistrically oserved.Ang-2 protein expression in the tissue surrounding the area of infarction was measured by Western blotting.VEGF expression in the MSCs increased after transfection at a rate of approximately 28%±3.4%.ELISA showed that the expression of VEGF in the cerebral tissue was significantly increased after induction of infarction,peaking on the 4th day and decreasing to the levels of the sham surgery group (normal) within 7 to 10 days.The VEGF level was significantly higher at each time point in the VEGF-MSC and MSC groups compared to the model group.Moreover,the VEGF level was higher in the VEGF-MSC group than in the MSC group and stayed relatively high until the 10th day.The immunohistochemical results showed that 10 days after the infarction,the number of Ang-2 and CD34-expressing cells in the area surrounding the infarction was significantly higher in the VEGF-MSC group and the MSC group compared to the model group.Moreover,the VEGF level was higher in the VEGF-MSC group than the MSC group.A similar trend in Ang-2 protein expression was revealed by Western blotting.In the MCAO rat model transfected with modified MSCs over-expressing VEGF,compared to the MSC transplantation group,the concentration of VEGF was significantly increased in the brain tissue after cerebral infarction.In addition,the level of Ang-2 was up-regulated,with angiogenesis promoted,the blood supply to the areas surrounding the cerebral infarction increased,and neurological function improved.We are led to speculate that the synergistic effects of VEGF and Ang-2 may be responsible for the angiogenesis following cerebral infarction.

Adipose-derived mesenchymal stromal/stem cells: An update on their phenotype in vivo and in vitro

Adipose tissue is a rich, ubiquitous and easily acces-sible source for multipotent stromal/stem cells and has, therefore, several advantages compared to other sourc-es of mesenchymal stromal/stem cells. Several studies have tried to identify the origin of the stromal/stem cell population within adipose tissue in situ. This is a complicated attempt because no marker has currently been described which unambiguously identifies native adipose-derived stromal/stem cells(ASCs). Isolated and cultured ASCs are a non-uniform preparation consisting of several subsets of stem and precursor cells. Cultured ASCs are characterized by their expression of a panel of markers(and the absence of others), whereas their in vitro phenotype is dynamic. Some markers were ex-pressed de novo during culture, the expression of some markers is lost. For a long time, CD34 expression was solely used to characterize haematopoietic stem and progenitor cells, but now it has become evident that it is also a potential marker to identify an ASC subpopula-tion in situ and after a short culture time. Nevertheless, long-term cultured ASCs do not express CD34, perhaps due to the artificial environment. This review gives an update of the recently published data on the origin and phenotype of ASCs both in vivo and in vitro. In addition, the composition of ASCs(or their subpopula-tions) seems to vary between different laboratories andpreparations. This heterogeneity of ASC preparationsmay result from different reasons. One of the main problems in comparing results from different laborato-ries is the lack of a standardized isolation and culture protocol for ASCs. Since many aspects of ASCs, suchas the differential potential or the current use in clinical trials, are fully described in other recent reviews, this review further updates the more basic research issues concerning ASCs' subpopulations, heterogeneity andculture standardization.

Mesenchymal stromal cells from human perinatal tissues: From biology to cell therapy

Cell-based regenerative medicine is of growing interest in biomedical research. The role of stem cells in this context is under intense scrutiny and may help to define principles of organ regeneration and develop innovative therapeutics for organ failure. Utilizing stem and progenitor cells for organ replacement has been conducted for many years when performing hematopoietic stem cell transplantation. Since the first successful transplantation of umbilical cord blood to treat hematological malignancies, non-hematopoietic stem and progenitor cell populations have recently been identified within umbilical cord blood and other perinatal and fetal tissues. A cell population entitled mesenchymal stromal cells (MSCs) emerged as one of the most intensely studied as it subsumes a variety of capacities: MSCs can differentiate into various subtypes of the mesodermal lineage, they secrete a large array of trophic factors suitable of recruiting endogenous repair processes and they are immunomodulatory.Focusing on perinatal tissues to isolate MSCs, we will discuss some of the challenges associated with these cell types concentrating on concepts of isolation and expansion, the comparison with cells derived from other tissue sources, regarding phenotype and differentiation capacity and finally their therapeutic potential.

Proliferation and phenotypic changes of stromal cells in response to varying estrogen/androgen levels in castrated rats

It is known that human benign prostatic hyperplasia might arise from an estrogen/androgen （E/T） imbalance. We studied the response of castrated rat prostate to different ratios of circulating E/T. The castrated male Wistar rats were randomly injected with E/T at different ratios for 4 weeks. The prostates of E/T （1：100） group showed a distinct prostatic hyperplasia response by prostatic index, hematoxylin and eosin staining, and quantitative immunohistochemical analysis of a-smooth muscle actin （SMA）. In this group, cells positive for Vimentin, non-muscle myosin heavy chain （NMMHC） and proliferating cell nuclear antigen （PCNA） increased in the stroma and epithelium. Furthermore, the mRNA levels of smooth muscle myosin heavy chain （SMMHC） and NMMHC increased. So E/T at a ratio of 1：100 can induce a stromal hyperplastic response in the prostate of castrated rats. The main change observed was an increase of smooth muscle cells, whereas some epithelial changes were also seen in the rat prostates.

Apoptosis during β-mercaptoethanol-induced differentiation of adult adipose-derived stromal cells into neurons

β-mercaptoethanol can induce adipose-derived stromal cells to rapidly and efficiently differentiate into neurons in vitro.However,because of the short survival time of the differentiated cells,clinical applications for this technique are limited.As such,we examined apoptosis of neurons differentiated from adipose-derived stromal cells induced with β-mercaptoethanol in vitro using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and transmission electron microscopy.The results revealed that the number of surviving cells decreased and apoptosis rate increased as induction time extended.Taken together,these results suggest that apoptosis occurring in the process of adipose-derived stromal cells differentiating into neurons is the main cause of cell death.However,the mechanism underlying cellular apoptosis should be researched further to develop methods of controlling apoptosis for clinical applications.

A novel ethanol-based method to induce differentiation of adipose-derived stromal cells into astrocytes

The quantity and survival time of astrocytes,which were differentiated from adult adipose-derived stromal cells after exposure to an inducer containing 3-isobutyl-1-methylxanthine,have thus far been unsatisfactory.The present study investigated the growth and differentiation characteristics of induced astrocytes by observing their growth curves.After induction for 48 hours with an inducer containing 0.5% ethanol,some adult adult adipose-derived stromal cells displayed typical astrocytic morphology.The cell quantity gradually decreased with prolonged induction time.Nestin,glial fibrillary acidic protein,and S-100 expression reached peak levels at 14 days,but neuron-specific enolase was not expressed.These results suggest that the induced astrocytes reached their peak at 14 days.Further optimization of the culture environment may yield mature astrocytes with normal functions,in greater quantity,and prolonged survival time.

Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative andadvantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine.