Stem Cell Tissue Regeneration Research Articles

Scale-Up Engineering Super-Energetic Adipose-Derived Mesenchymal Stem Cells with Mitochondrial Transplantation for Tissue Regeneration

Stem cell therapies are unsatisfactory due to poor cell survival and engraftment. Stem cell used for therapy must be properly “tuned” for a harsh in vivo environment. Herein, we report that transfer of exogenous mitochondria (mito) to adipose-derived mesenchymal stem cells (ADSCs) can effectively boost their energy levels, enabling efficient cell engraftment. Importantly, the entire process of exogeneous mitochondrial endocytosis is captured by high-content live-cell imaging. Mitochondrial transfer leads to acutely enhanced bioenergetics, with nearly 17% of higher adenosine 5′-triphosphate (ATP) levels in ADSCs treated with high mitochondrial dosage, and further results in altered secretome profiles of ADSCs. Mitochondrial transfer also induced the expression of 334 mRNAs in ADSCs which are mainly involved in signaling pathways related to DNA replication and cell cycle. We propose that increase in ATP and cyclin-dependent kinase (CDK) 1 and 2 expression might be responsible for promoting enhanced proliferation, migration and differentiation of ADSCs in vitro. More importantly, mito-transferred ADSCs display prolonged cell survival, engraftment and horizontal transfer of exogenous mitochondria to surrounding cells in a full-thickness skin defect rat model with improved skin repair compared with non-treated ADSCs. These results demonstrate that intracellular mitochondrial transplantation is a promising strategy to engineer stem cells for tissue regeneration.

ZIF-8 Modified Polypropylene Membrane: A Biomimetic Cell Culture Platform with a View to the Improvement of Guided Bone Regeneration.

PurposeDespite the significant advances in modeling of biomechanical aspects of cell microenvironment, it remains a major challenge to precisely mimic the physiological condition of the particular cell niche. Here, the metal–organic frameworks (MOFs) have been introduced as a feasible platform for multifactorial control of cell-substrate interaction, given the wide range of physical and mechanical properties of MOF materials and their structural flexibility.ResultsIn situ crystallization of zeolitic imidazolate framework-8 (ZIF-8) on the polydopamine (PDA)-modified membrane significantly raised surface energy, wettability, roughness, and stiffness of the substrate. This modulation led to an almost twofold increment in the primary attachment of dental pulp stem cells (DPSCs) compare to conventional plastic culture dishes. The findings indicate that polypropylene (PP) membrane modified by PDA/ZIF-8 coating effectively supports the growth and proliferation of DPSCs at a substantial rate. Further analysis also displayed the exaggerated multilineage differentiation of DPSCs with amplified level of autocrine cell fate determination signals, like BSP1, BMP2, PPARG, FABP4, ACAN, and COL2A. Notably, osteogenic markers were dramatically overexpressed (more than 100-folds rather than tissue culture plate) in response to biomechanical characteristics of the ZIF-8 layer.ConclusionHence, surface modification of cell culture platforms with MOF nanostructures proposed as a powerful nanomedical approach for selectively guiding stem cells for tissue regeneration. In particular, PP/PDA/ZIF-8 membrane presented ideal characteristics for using as a barrier membrane for guided bone regeneration (GBR) in periodontal tissue engineering.

Advanced Biomaterials and Techniques for Oral Tissue Engineering and Regeneration-A Review.

The reconstruction or repair of oral and maxillofacial functionalities and aesthetics is a priority for patients affected by tooth loss, congenital defects, trauma deformities, or various dental diseases. Therefore, in dental medicine, tissue reconstruction represents a major interest in oral and maxillofacial surgery, periodontics, orthodontics, endodontics, and even daily clinical practice. The current clinical approaches involve a vast array of techniques ranging from the traditional use of tissue grafts to the most innovative regenerative procedures, such as tissue engineering. In recent decades, a wide range of both artificial and natural biomaterials and scaffolds, genes, stem cells isolated from the mouth area (dental follicle, deciduous teeth, periodontal ligament, dental pulp, salivary glands, and adipose tissue), and various growth factors have been tested in tissue engineering approaches in dentistry, with many being proven successful. However, to fully eliminate the problems of traditional bone and tissue reconstruction in dentistry, continuous research is needed. Based on a recent literature review, this paper creates a picture of current innovative strategies applying dental stem cells for tissue regeneration in different dental fields and maxillofacial surgery, and offers detailed information regarding the available scientific data and practical applications.

Human Mesenchymal Stem Cell Derived Exosomes Enhance Cell-Free Bone Regeneration by Altering Their miRNAs Profiles.

Implantation of stem cells for tissue regeneration faces significant challenges such as immune rejection and teratoma formation. Cell‐free tissue regeneration thus has a potential to avoid these problems. Stem cell derived exosomes do not cause immune rejection or generate malignant tumors. Here, exosomes that can induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) are identified and used to decorate 3D‐printed titanium alloy scaffolds to achieve cell‐free bone regeneration. Specifically, the exosomes secreted by hMSCs osteogenically pre‐differentiated for different times are used to induce the osteogenesis of hMSCs. It is discovered that pre‐differentiation for 10 and 15 days leads to the production of osteogenic exosomes. The purified exosomes are then loaded into the scaffolds. It is found that the cell‐free exosome‐coated scaffolds regenerate bone tissue as efficiently as hMSC‐seeded exosome‐free scaffolds within 12 weeks. RNA‐sequencing suggests that the osteogenic exosomes induce the osteogenic differentiation by using their cargos, including upregulated osteogenic miRNAs (Hsa‐miR‐146a‐5p, Hsa‐miR‐503‐5p, Hsa‐miR‐483‐3p, and Hsa‐miR‐129‐5p) or downregulated anti‐osteogenic miRNAs (Hsa‐miR‐32‐5p, Hsa‐miR‐133a‐3p, and Hsa‐miR‐204‐5p), to activate the PI3K/Akt and MAPK signaling pathways. Consequently, identification of osteogenic exosomes secreted by pre‐differentiated stem cells and the use of them to replace stem cells represent a novel cell‐free bone regeneration strategy.

Decellularized adipose matrix provides an inductive microenvironment for stem cells in tissue regeneration.

Stem cells play a key role in tissue regeneration due to their self-renewal and multidirectional differentiation, which are continuously regulated by signals from the extracellular matrix (ECM) microenvironment. Therefore, the unique biological and physical characteristics of the ECM are important determinants of stem cell behavior. Although the acellular ECM of specific tissues and organs (such as the skin, heart, cartilage, and lung) can mimic the natural microenvironment required for stem cell differentiation, the lack of donor sources restricts their development. With the rapid development of adipose tissue engineering, decellularized adipose matrix (DAM) has attracted much attention due to its wide range of sources and good regeneration capacity. Protocols for DAM preparation involve various physical, chemical, and biological methods. Different combinations of these methods may have different impacts on the structure and composition of DAM, which in turn interfere with the growth and differentiation of stem cells. This is a narrative review about DAM. We summarize the methods for decellularizing and sterilizing adipose tissue, and the impact of these methods on the biological and physical properties of DAM. In addition, we also analyze the application of different forms of DAM with or without stem cells in tissue regeneration (such as adipose tissue), repair (such as wounds, cartilage, bone, and nerves), in vitro bionic systems, clinical trials, and other disease research.

Biological Effects of Tricalcium Silicate Nanoparticle-Containing Cement on Stem Cells from Human Exfoliated Deciduous Teeth.

Nanomaterials can enhance interactions with stem cells for tissue regeneration. This study aimed to investigate the biological effects of tricalcium silicate nanoparticle-containing cement (Biodentine™) during or after setting on stem cells from human exfoliated deciduous teeth (SHED) to mimic clinically relevant situations in which materials are adapted. Specimens were divided into four groups depending on the start of extraction time (during (3, 6 and 12 min) or after setting (24 h)) and extracted in culture medium for 24 h for further physicochemical and biological analysis. After cell viability in serially diluted extracts was evaluated, odontogenic differentiation on SHED was evaluated by ARS staining using nontoxic conditions. A physicochemical analysis of extracts or specimens indicated different Ca ion content, pH, and surface chemistry among groups, supporting the possibility of different biological functionalities depending on the extraction starting conditions. Compared to the ‘after setting’ group, all ‘during setting’ groups showed cytotoxicity on SHED. The during setting groups induced more odontogenic differentiation at the nontoxic concentrations compared to the control. Thus, under clinically simulated extract conditions at nontoxic concentrations, Biodentine™ seemed to be a promising odontoblast differentiating biomaterial that is helpful for dental tissue regeneration. In addition, to simulate clinical situations when nanoparticle-containing cement is adjusted, biological effects during setting need to be considered.

Selected Nanomaterials' Application Enhanced with the Use of Stem Cells in Acceleration of Alveolar Bone Regeneration during Augmentation Process.

Regenerative properties are different in every human tissue. Nowadays, with the increasing popularity of dental implants, bone regenerative procedures called augmentations are sometimes crucial in order to perform a successful dental procedure. Tissue engineering allows for controlled growth of alveolar and periodontal tissues, with use of scaffolds, cells, and signalling molecules. By modulating the patient’s tissues, it can positively influence poor integration and healing, resulting in repeated implant surgeries. Application of nanomaterials and stem cells in tissue regeneration is a newly developing field, with great potential for maxillofacial bony defects. Nanostructured scaffolds provide a closer structural support with natural bone, while stem cells allow bony tissue regeneration in places when a certain volume of bone is crucial to perform a successful implantation. Several types of selected nanomaterials and stem cells were discussed in this study. Their use has a high impact on the efficacy of the current and future procedures, which are still challenging for medicine. There are many factors that can influence the regenerative process, while its general complexity makes the whole process even harder to control. The aim of this study was to evaluate the effectiveness and advantage of both stem cells and nanomaterials in order to better understand their function in regeneration of bone tissue in oral cavity.

Identification of Human Very Small Embryonic like Stem Cells (VSELS) in Human Heart Tissue Among Young and Old Individuals.

Very Small Embryonic-Like (VSEL) stem cells are a proposed pluripotent population, residing in adult tissues. VSELs have been described in multiple tissues including bone marrow, cord blood, and gonads. They exhibit multiple characteristics of embryonic stem cells including the ability to differentiate into cellular lineages of all three germ layers, including cardiomyocytes and vascular endothelial cells. However, their presence in adult solid organs such as heart in humans has not been established. VSELs are valuable source of stem cells for tissue regeneration and replacement of cells for turnover and usual wear-and-tear. The purpose of our study was to explore the existence of human VSELs (huVSELs) in human heart tissue and examine the changes in their prevalence with aging and cardiac disease. Human heart tissue, collected from healthy and ischemic heart disease subjects was examined for the prevalence of VSELS, defined as CD45-/CD133+/SSEA4+. Both epicardial and endocardial tissues were examined comparing VSEL numbers across different age groups. Our data confirm the existence of huVSELs in adult hearts with decreasing prevalence during aging. This is the first evidence of huVSELs in adult cardiac tissue. Cardiac huVSELs could be further explored in future studies to characterize their primitive potential and therapeutic potential in regenerative studies.

Biomaterials for Personalized Cell Therapy.

Cell therapy has already had an important impact on healthcare and provided new treatments for previously intractable diseases. Notable examples include mesenchymal stem cells for tissue regeneration, islet transplantation for diabetes treatment, and T cell delivery for cancer immunotherapy. Biomaterials have the potential to extend the therapeutic impact of cell therapies by serving as carriers that provide 3D organization and support cell viability and function. With the growing emphasis on personalized medicine, cell therapies hold great potential for their ability to sense and respond to the biology of an individual patient. These therapies can be further personalized through the use of patient-specific cells or with precision biomaterials to guide cellular activity in response to the needs of each patient. Here, the role of biomaterials for applications in tissue regeneration, therapeutic protein delivery, and cancer immunotherapy is reviewed, with a focus on progress in engineering material properties and functionalities for personalized cell therapies.

An Overview on Stem Cells in Tissue Regeneration

Deteriorations in tissues and decline in organ functions, due to chronic diseases or with advancing age or sometimes due to infections or injuries, can severely compromise the quality of life of an individual. Regenerative medicine, a field of medical research focuses on replacing non-functional or dead cells or repairing or regenerating tissues and organs to restore normal functions of an impaired organ. Approaches used in regenerative therapy for achieving the objective employ a number of means which include soluble biomolecules, stem cell transplants, tissue engineering, gene therapy and reprogramming of cells according to target tissue types. Stem cells transplant and tissue regeneration methods for treating various diseases have rapidly grown in usage over the past decades or so. There are different types of stem cells such as mesenchymal, hematopoietic, embryonic, mammary, intestinal, endothelial, neural, olfactory, neural crest, testicular and induced pluripotent stem cells. This review covers the recent advances in tissue regeneration and highlights the application of stem cell transplants in treating many life-threatening diseases or in improving quality of life. Remarkable progress in stem cell research has established that the cell-based therapy could be an option for treating diseases which could not be cured by conventional medical means till recent. Stem cells play major roles in regenerative medicine with its exceptional characteristics of self-renewal capacity and potential to differentiate into almost all types of cells of a body. Vast number of reports on preclinical and clinical application of stem cells revealed its vital role in disease management and many pharmacological industries around the globe working to achieve effective stem cell based products.

Application of Tonsil-Derived Mesenchymal Stem Cells in Tissue Regeneration: Concise Review.

Since the discovery of stem cells and multipotency characteristics of mesenchymal stem cells (MSCs), there has been tremendous development in regenerative medicine. MSCs derived from bone marrow have been widely used in various research applications, yet there are limitations such as invasiveness of obtaining samples, low yield and proliferation rate, and questions regarding their practicality in clinical applications. Some have suggested that MSCs from other sources, specifically those derived from palatine tonsil tissues, that is, tonsil‐derived MSCs (TMSCs), could be considered as a new potential therapeutic tool in regenerative medicine due to their superior proliferation rate and differentiation capabilities with low immunogenicity and ease of obtaining. Several studies have determined that TMSCs have differentiation potential not only into the mesodermal lineage but also into the endodermal as well as ectodermal lineages, expanding their potential usage and placing them as an appealing option to consider for future studies in regenerative medicine. In this review, the differentiation capacities of TMSCs and their therapeutic competencies from past studies are addressed. stem cells 2019;37:1252–1260

Recent Progress in the Biological Basis of Remodeling Tissue Regeneration Using Nanofibers: Role of Mesenchymal Stem Cells and Biological Molecules

Opening up new and unlimited avenues in the biomedical field, tissue engineering and regenerative medicine, the electrospinning process is considered as a versatile and the most preferred technique for the fabrication of nanofibers. These tailor-designed nanofibers provide a desirable and bio-inspired physiological niche to cells for better attachment and subsequent proliferation. In this review, an attempt is made to explain the importance of various topological and morphological parameters of nanofibrous scaffolds for efficient bio-mimicking. Some novel approaches (e.g., appropriate functionalization and extracellular matrix derived from decellularization) utilized for better mimicking and exponential growth of regenerating tissues are also discussed. Furthermore, this review highlights the important parameters necessary for the attachment, proliferation and differentiation of the mesenchymal stem cells for tissue regeneration. The importance of growth factors and their role after introducing the electrospinning techniques for efficient delivery and their role in the proliferation of mesenchymal stem cells in the different specific lineage (e.g., tenogenic, chondrogenic, neurogenic and osteogenic differentiation) are discussed.

MiRNA-21 promotes osteogenesis via the PTEN/PI3K/Akt/HIF-1\u03b1 pathway and enhances bone regeneration in critical size defects

BackgroundFunctional reconstruction of maxillofacial bone defects is a considerable clinical challenge. Many studies have emphasized the osteogenic and angiopoietic abilities of stem cells for tissue regeneration. We previously showed that microRNA-21 (miRNA-21) can promote angiogenesis in human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs). In the present study, the role of miRNA-21 in osteogenic differentiation of bone marrow-derived stem cells (BMSCs) was investigated.MethodsWestern blotting and qPCR were performed to investigate the influences of miRNA-21 on osteogenic differentiation of BMSCs. The effects of miRNA-21 on PTEN/PI3K/Akt/HIF-1α pathway were also assessed using western blotting. To further evaluate the roles of miRNA-21 in osteogenesis in vivo, we conducted animal experiments in rat and canine. New bone formation was assessed using micro-CT and histological methods.ResultsIn the present study, we found that miRNA-21 promotes the migration and osteogenic differentiation of bone marrow-derived stem cells (BMSCs) in vitro. Using gain- and loss-of-function studies, we found that miRNA-21 promoted the osteogenic ability of BMSCs by increasing P-Akt and HIF-1α activation. Finally, we verified the essential role of miRNA-21 in osteogenesis by implanting a miRNA-21-modified BMSCs/β-tricalcium phosphate (β-TCP) composite into critical size defects. Radiography, micro-CT, and histology revealed significantly greater volume of new bone formation in the miRNA-21 group than in the control group.ConclusionIn conclusion, our study demonstrated an essential role of miRNA-21 in promoting maxillofacial bone regeneration via the PTEN/PI3K/Akt/HIF-1α pathway.

Innovative Biomaterials for Bone Regrowth.

The regenerative medicine, a new discipline that merges biological sciences and the fundamental of engineering to develop biological substitutes, has greatly benefited from recent advances in the material engineering and the role of stem cells in tissue regeneration. Regenerative medicine strategies, involving the combination of biomaterials/scaffolds, cells, and bioactive agents, have been of great interest especially for the repair of damaged bone and bone regrowth. In the last few years, the life expectancy of our population has progressively increased. Aging has highlighted the need for intervention on human bone with biocompatible materials that show high performance for the regeneration of the bone, efficiently and in a short time. In this review, the different aspects of tissue engineering applied to bone engineering were taken into consideration. The first part of this review introduces the bone cellular biology/molecular genetics. Data on biomaterials, stem cells, and specific growth factors for the bone regrowth are reported in this review.

Roles of Mesenchymal Stem Cells in Tissue Regeneration and Immunomodulation.

Mesenchymal stem cells are classified as multipotent stem cells, due to their capability to transdifferentiate into various lineages that develop from mesoderm. Their popular appeal as cell-based therapy was initially based on the idea of their ability to restore tissue because of their differentiation potential in vitro; however, the lack of evidence of their differentiation to target cells in vivo led researchers to focus on their secreted trophic factors and their role as potential powerhouses on regulation of factors under different immunological environments and recover homeostasis. To date there are more than 800 clinical trials on humans related to MSCs as therapy, not to mention that in animals is actively being applied as therapeutic resource, though it has not been officially approved as one. But just as how results from clinical trials are important, so is to reveal the biological mechanisms involved on how these cells exert their healing properties to further enhance the application of MSCs on potential patients. In this review, we describe characteristics of MSCs, evaluate their benefits as tissue regenerative therapy and combination therapy, as well as their immunological properties, activation of MSCs that dictate their secreted factors, interactions with other immune cells, such as T cells and possible mechanisms and pathways involved in these interactions.

Biology of teeth and implants: Host factors - pathology, regeneration, and the role of stem cells.

In chronic periodontitis and peri-implantitis, cells of the innate and adaptive immune systems are involved directly in the lesions within the tissues of the patient. Absence of a periodontal ligament around implants does not prevent a biologic process similar to that of periodontitis from affecting osseointegration. Our first focus is on factors in the biology of individuals that are responsible for the susceptibility of such individuals to chronic periodontitis and to peri-implantitis. Genetic factors are of significant importance in susceptibility to these diseases. Genetic factors of the host affect the composition of the oral microbiome in the same manner that they influence other microbiomes, such as those of the intestines and of the lungs. Our second focus is on the central role of stem cells in tissue regeneration, in the functioning of innate and adaptive immune systems, and in metabolism of bone. Epithelial cell rests of Malassez (ERM) are stem cells of epithelial origin that maintain the periodontal ligament as well as the cementum and alveolar bone associated with the ligament. The tissue niche within which ERM are found extends into the supracrestal areas of collagen fiber-containing tissues of the gingivae above the bony alveolar crest. Maintenance and regeneration of all periodontal tissues involves the activity of a variety of stem cells. The success of dental implants indicates that important groups of stem cells in the periodontium are active to enable that biologic success. Successful replantation of avulsed teeth and auto-transplantation of teeth is comparable to placing dental implants, and so must also involve periodontal stem cells. Biology of teeth and biology of implants represents the biology of the various stem cells that inhabit specialized niches within the periodontal tissues. Diverse biologic processes must function together successfully to maintain periodontal health. Osseointegration of dental implants does not involve formation of cementum or collagen fibers inserted into cementum - indicating that some stem cells are not active around dental implants or their niches are not available. Investigation of these similarities and differences between teeth and implants will help to develop a better understanding of the biology and physiologic functioning of the periodontium.

Recent Progress in Stem Cell Modification for Cardiac Regeneration.

During the past decades, stem cell-based therapy has acquired a promising role in regenerative medicine. The application of novel cell therapeutics for the treatment of cardiovascular diseases could potentially achieve the ambitious aim of effective cardiac regeneration. Despite the highly positive results from preclinical studies, data from phase I/II clinical trials are inconsistent and the improvement of cardiac remodeling and heart performance was found to be quite limited. The major issues which cardiac stem cell therapy is facing include inefficient cell delivery to the site of injury, accompanied by low cell retention and weak effectiveness of remaining stem cells in tissue regeneration. According to preclinical and clinical studies, various stem cells (adult stem cells, embryonic stem cells, and induced pluripotent stem cells) represent the most promising cell types so far. Beside the selection of the appropriate cell type, researchers have developed several strategies to produce “second-generation” stem cell products with improved regenerative capacity. Genetic and nongenetic modifications, chemical and physical preconditioning, and the application of biomaterials were found to significantly enhance the regenerative capacity of transplanted stem cells. In this review, we will give an overview of the recent developments in stem cell engineering with the goal to facilitate stem cell delivery and to promote their cardiac regenerative activity.

A Novel Method to Induce Cartilage Regeneration with Cubic Microcartilage

Cartilage tissue is characterized by its poor regenerative properties, and the clinical performance of cartilage grafts to replace cartilage defects has been unsatisfactory. Recently, cartilage regeneration with mature chondrocytes and stem cells has been developed and applied in clinical settings. However, there are challenges with the use of mature chondrocytes and stem cells for tissue regeneration, including the high costs associated with the standard stem cell isolation methods and the decreased cell viability due to cell manipulation. Previous studies demonstrated that cartilage can be regenerated from chondrocyte clusters that contain stem cells. Based upon some of the existing techniques, the goal of this study was to develop a novel and practical method to induce cartilage regeneration. A microslicer device was developed to process cartilage tissues into micron-size cartilage (microcartilage) in a minimally invasive manner. We evaluated microcartilage sizes and demonstrated 100-400 µm as optimal for generating a high cell yield with collagenase digestion. In addition, autologous intrafascial implantation of the composites of microcartilage and an absorbable scaffold with a slow-release system of basic fibroblast growth factor (bFGF) was carried out to induce cartilage regeneration. Our results demonstrated that the extent of bFGF diffusion depends on the size of microcartilage, and that cartilage regeneration was induced most effectively with 100 µm of microcartilage via SOX5 upregulation. These findings suggest that cartilage regeneration is possible with microcartilage as a source of cells without ex vivo cell expansion.

Amniotic Fluid Stem Cells: A New Era in Regenerative Medicine.

Regenerative medicine has become an emerging field which focuses on repair, replacement or regeneration of cells, tissues and the entire organs. The regeneration may occur in patient's own body by using their system as a bioreactor, e.g., cell therapy that involves transplantation of stem cells capable of proliferating, differentiating and replacing damaged host cells. As the field of regenerative medicine advances, and sources of stem cells has been intensified. Though embryonic and adult tissues can be used for isolation of pluripotent stem cells, the amniotic fluid (AF) has been proposed as an alternative source of stem cells for tissue regeneration. AF cells could be banked and used for either allogeneic or autologous transplantation.

Osteogenic Potential of Adipose-Derived Macrospheroids Cocultured with CD11b+ Monocytes.

Among potential cell-based therapies, adipose-derived stem cells (ASCs) have been proposed as a promising source of stem cells for tissue regeneration. Although many recent clinical trials have investigated the use of adipose tissue or ASCs in transplantation, analysis of the microstructures of outgrowing macrosized spheroids (macrospheroids) or three-dimensional coculture of ASC spheroids and monocyte/macrophage lineages has not been performed. The aim of this study was to analyze the microstructures of murine-derived ASC macrospheroids and the growth and osteogenic potential of these macrospheroids in a three-dimensional environment and after calcification induction by coculture with monocytes. The histologic structures of murine-derived ASC macrospheroids and the expression of marker genes for multipotency within these macrospheroids were analyzed by hematoxylin and eosin staining and in situ hybridization. ASC macrospheroid microstructures were observed by transmission electron microscopy, and cell proliferation in the spheroids was analyzed. Additionally, the growth and osteogenic potential of these macrospheroids were assessed in two-dimensional and three-dimensional environments and after calcification induction by coculture with monocytes. The expression of Oct3/4, Nanog, and Sox2 was detected even in the deep zone of spheroids, although higher expression was observed at the surface. Cell proliferation was detected within the spheroid centers. Observation of spheroid microstructure revealed extracellular matrix production within the spheroid architecture. Transplantation of a spheroid on the hydroxyapatite disc resulted in three-dimensional cell growth, filling the disc. Coculture of the spheroids with monocytes led to the formation of many osteoclast-like, multinucleated cells, and calcification was observed after 3 weeks of coculture. ASC spheroids exhibited high capacity for dynamic three-dimensional growth and osteogenic differentiation. Furthermore, ASC spheroids promoted monocyte differentiation into osteoclast-like cells, which may enhance the osteogenic potential of ASC spheroids.