Computer-aided exploration of multiobjective optimal temperature profiles in slow freezing for human induced pluripotent stem cells.

Human induced pluripotent stem (hiPS) cells have demonstrated promising potential in regenerative medical therapeutics. After successful clinical trials, the demand for hiPS cells has steadily increased. Therefore, the optimization of hiPS cell freezing processes for storage and transportation is essential. Here, we presented a computer-aided exploration of multiobjective optimal temperature profiles in slow freezing for hiPS cells. This study was based on a model that calculates cell survival rates after thawing, and the model was extended to evaluate cell potentials until 24 h after seeding. To estimate parameter values for this extension, freezing experiments were performed using constant cooling rates. Using quality and productivity indicators, we evaluated 16,206 temperature profiles using our model, and a promising profile was obtained. Finally, an experimental investigation of the profile was undertaken, and the contribution of the temperature profile to both quality and productivity was confirmed.

A Kpna1-deficient psychotropic drug-induced schizophrenia model mouse for studying gene-environment interactions.

KPNA1 is a mediator of nucleocytoplasmic transport that is abundantly expressed in the mammalian brain and regulates neuronal differentiation and synaptic function. De novo mutations in Kpna1 have been identified using genome-wide association studies in humans with schizophrenia; however, it remains unclear how KPNA1 contributes to schizophrenia pathogenesis. Recent studies have suggested a complex combination of genetic and environmental factors that are closely related to psychiatric disorders. Here, we found that subchronic administration of phencyclidine, a psychotropic drug, induced vulnerability and behavioral abnormalities consistent with the symptoms of schizophrenia in Kpna1-deficient mice. Microarray assessment revealed that the expression levels of dopamine d1/d2 receptors, an RNA editing enzyme, and a cytoplasmic dynein component were significantly altered in the nucleus accumbens brain region in a gene-environment (G × E) interaction-dependent manner. Our findings demonstrate that Kpna1-deficient mice may be useful as a G × E interaction mouse model for psychiatric disorders and for further investigation into the pathogenesis of such diseases and disorders.

A simple tool for the synchronous differentiation of human induced pluripotent stem cells into pancreatic progenitors.

Efficient differentiation of human induced pluripotent stem cells (hiPSCs) into functional pancreatic cells holds great promise for diabetes research and treatment. However, a robust culture strategy for producing pancreatic progenitors with high homogeneity is lacking. Here, we established a simple differentiation strategy for generating synchronous iPSC-derived pancreatic progenitors via a two-step method of sequential cell synchronization using botulinum hemagglutinin (HA), an E-cadherin function-blocking agent. Of the various methods tested, the first-step synchronization method with HA exposure induces a synchronous switch from E- to N-cadherin and N- to E-cadherin expression by spatially controlling heterogeneous cell distribution, subsequently improving their competency for directed differentiation into definitive endodermal cells from iPSCs. The iPSC-derived definitive endodermal cells can efficiently generate PDX1+ and NKX6.1+ pancreatic progenitor cells in high yields. The PDX1+ and PDX1+ /NKX6.1+ cell densities showed 1.6- and 2.2-fold increases, respectively, compared with those from unsynchronized cultures. The intra-run and inter-run coefficient of variation were below 10%, indicating stable and robust differentiation across different cultures and runs. Our approach is a simple and efficient strategy to produce large quantities of differentiated cells with the highest homogeneity during multistage pancreatic progenitor differentiation, providing a potential tool for guided differentiation of iPSCs to functional insulin-producing cells.

Role of glycogen synthase kinase-3ß in dependence and abuse liability of alcohol.

BACKGROUND: Alcohol is a major abused drug worldwide that contributes substantially to health and social problems. These problems result from acute alcohol overuse as well as chronic use, leading to alcohol use disorder (AUD). A major goal of this field is to establish a treatment for alcohol abuse and dependence in patients with AUD. The central molecular mechanisms of acute alcohol actions have been extensively investigated in rodent models. AIMS: One of the central mechanisms that may be involved is glycogen synthase kinase-3ß (GSK-3ß) activity, a key enzyme involved in glycogen metabolism but which has crucial roles in numerous cellular processes. Although the exact mechanisms leading from acute alcohol actions to these chronic changes in GSK-3ß function are not yet clear, GSK-3ß nonetheless constitutes a potential therapeutic target for AUD by reducing its function using GSK-3ß inhibitors. This review is focused on the correlation between GSK-3ß activity and the degree of alcohol consumption. METHODS: Research articles regarding investigation of effect of GSK-3ß on alcohol consumption in rodents were searched on PubMed, Embase, and Scopus databases using keywords "glycogen synthase kinase," "alcohol (or ethanol)," "intake (or consumption)," and evaluated by changes in ratios of pGSK-3ßSer9/pGSK-3ß. RESULTS: In animal experiments, GSK-3ß activity decreases in the brain under forced and voluntary alcohol consumption while GSK-3ß activity increases under alcohol-seeking behavior. CONCLUSIONS: Several pieces of evidence suggest that alterations in GSK-3ß function are important mediators of chronic ethanol actions, including those related to alcohol dependence and the adverse effects of chronic ethanol exposure.

Growth prolongation of human induced pluripotent stem cell aggregate in three-dimensional suspension culture system by addition of botulinum hemagglutinin.

Human induced pluripotent stem cells (hiPSCs) can be used in regenerative therapy as an irresistible cell source, and so the development of scalable production of hiPSCs for three-dimensional (3D) suspension culture is required. In this study, we established a simple culture strategy for improving hiPSC aggregate growth using botulinum hemagglutinin (HA), which disrupts cell-cell adhesion mediated by E-cadherin. When HA was added to the suspension culture of hiPSC aggregates, E-cadherin-mediated cell-cell adhesion was temporarily disrupted within 24 h, but then recovered. Phosphorylated myosin light chain, a contractile force marker, was also recovered at the periphery of hiPSC aggregates. The cell aggregates were suppressed the formation of collagen type I shell-like structures at the periphery by HA and collagen type I was homogenously distributed within the cell aggregates. In addition, these cell aggregates retained the proliferation marker Ki-67 throughout the cell aggregates. The apparent specific growth rate with HA addition was maintained continuously throughout the culture, and the final cell density was 1.7-fold higher than that in the control culture. These cells retained high expression levels of pluripotency markers. These observations indicated that relaxation of cell-cell adhesions by HA addition induced rearrangement of the mechanical tensions generated by actomyosin in hiPSC aggregates and suppression of collagen type I shell-like structure formation. These results suggest that this simple and readily culture strategy is a potentially useful tool for improving the scalable production of hiPSCs for 3D suspension cultures.

Agent-based approach for elucidating the release from collective arrest of cell motion in corneal epithelial cell sheet.

Changes in cell fluidity have been observed in various cellular tissues and are strongly linked to biological phenomena such as self-organization. Recent studies suggested variety of mechanisms and factors, which are still being investigated. This study aimed to investigate changes in cell fluidity in multi-layered cell sheets, by exploring the collective arrest of cell motion and its release in cultures of corneal epithelial cells. We constructed mathematical models to simulate the behaviors of individual cells, including cell differentiation and time-dependent changes in cell-cell connections, which are defined by stochastic or kinetic rules. Changes in cell fluidity and cell sheet structures were expressed by simulating autonomous cell behaviors and interactions in tissues using an agent-based model. A single-cell level spatiotemporal analysis of cell state transition between migratable and non-migratable states revealed that the release from collective arrest of cell motion was initially triggered by a decreased ability to form cell-cell connections in the suprabasal layers, and was propagated by chain migration. Notably, the disruption of cell-cell connections and stratification occurred in the region of migratable state cells. Hence, a modeling approach that considers time-dependent changes in cell properties and behavior, and spatiotemporal analysis at the single-cell level can effectively delineate emergent phenomena arising from the complex interplay of cells.

Effect of morphological change on the maturation of human induced pluripotent stem cell-derived cardiac tissue in rotating flow culture.

Introduction: Understanding the critical factors for the maturation of human induced pluripotent stem cell (hiPSC)-derived cardiac tissue is important for further development of culture techniques. Rotating flow culture, where the tissues float in the culture medium by balancing its gravitational settling and the medium flow generated in rotating disk-shaped culture vessels, is one of culture systems used for tissue engineering. It has previously been demonstrated that rotating flow culture leads to the formation of matured cardiac tissue with higher levels of function and structure than the other culture systems. However, the detailed mechanisms underlying the maturation of cardiac tissue remain unclear. This study investigated the maturation process of hiPSC-derived cardiac tissue in rotating flow culture with a focus on morphological changes in the tissue, which is a trigger for maturation. Methods: The cardiac tissue, which consisted of cardiomyocytes derived from hiPSCs, was cultured on the 3D scaffold of poly (lactic-co-glycolic) acid (PLGA)-aligned nanofibers, in rotating flow culture for 5 days. During the culture, the time profile of projected area of tissue and formation of maturation marker proteins (ß-myosin heavy chain and Connexin-43), tissue structure, and formation of nuclear lamina proteins (Lamin A/C) were compared with that in static suspension culture. Results: The ratio of the projected area of tissue significantly decreased from Day 0 to Day 3 due to tissue shrinkage. In contrast, Western blot analysis revealed that maturation protein markers of cardiomyocytes significantly increased after Day 3. In addition, in rotating flow culture, flat-shaped nuclei and fiber-like cytoskeletal structures were distributed in the surface region of tissue where medium flow was continuously applied. Moreover, Lamin A/C, which are generally formed in differentiated cells owing to mechanical force across the cytoskeleton and critically affect the maturation of cardiomyocytes, were significantly formed in the tissue of rotating flow culture. Conclusions: In this study, we found that spatial heterogeneity of tissue structure and tissue shrinkage occurred in rotating flow culture, which was not observed in static suspension culture. Moreover, from the quantitative analysis, it was also suggested that tissue shrinkage in rotating flow culture contributed its following tissue maturation. These findings showed one of the important characteristics of rotating flow culture which was not revealed in previous studies.

An in vitro culture platform to study the extracellular matrix remodeling potential of human mesenchymal stem cells.

The ability of mesenchymal stem cells (MSCs) to synthesize and degrade extracellular matrix (ECM) is important for MSC-based therapies. However, the therapeutic effects associated with ECM remodeling in cultured MSCs have been limited by the lack of a method to assess the ability of cultured cells to degrade ECM in vitro. Here, we describe a simple in vitro culture platform for studying the ECM remodeling potential of cultured MSCs using a high-density collagen (CL) surface. Cells on the CL surface have remarkable ability to degrade collagen fibrils by secreting matrix metalloproteinase (MMP); to study this, the marker collagen hybridizing peptide (CHP) was used. Confirming the ECM remodeling potential of MSCs with different population doublings (PDs), young and healthy γ-H2AX-negative cells, a marker of DNA damage and senescence, showed more extensive collagen degradation on the CL surface, whereas damaged cells of γ-H2AX-positive cells showed no collagen degradation. The frequency of γ-H2AX-/CHP + cells at PD = 0 was 49%, which was 4.9-fold higher than that at PD=13.07, whereas the frequency of γ-H2AX+/CHP- at PD=13.07 was 50%, which was 6.4-folds higher than that at PD=0. Further experimentation examining the in vitro priming effect of MSCs with the pro-inflammatory cytokine interferon-γ treatment showed increased frequency of cells with ECM remodeling potential with higher MMP secretion. Thus, this culture surface can be used for studying the ECM remodeling capacity of ex vivo-expanded MSCs in vitro and may serve as a platform for prediction in vivo ECM remodeling effect. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) remodeling potential of cultured mesenchymal stem cells (MSCs) is important for assessing the effectiveness of MSC-based therapy. However, methods to assess the ability of cultured cells to degrade ECM in vitro are still lacking. Here, we developed a simple in vitro culture platform to study the ECM remodeling potential of cultured MSCs using high-density collagen surfaces. This platform was used to evaluate the ECM remodeling potential of long-term ex vivo-expanded MSCs in vitro.

Phase-separated nuclear bodies of nucleoporin fusions promote condensation of MLL1/CRM1 and rearrangement of 3D genome structure.

NUP98 and NUP214 form chimeric fusion proteins that assemble into phase-separated nuclear bodies containing CRM1, a nuclear export receptor. However, these nuclear bodies' function in controlling gene expression remains elusive. Here, we demonstrate that the nuclear bodies of NUP98::HOXA9 and SET::NUP214 promote the condensation of mixed lineage leukemia 1 (MLL1), a histone methyltransferase essential for the maintenance of HOX gene expression. These nuclear bodies are robustly associated with MLL1/CRM1 and co-localized on chromatin. Furthermore, whole-genome chromatin-conformation capture analysis reveals that NUP98::HOXA9 induces a drastic alteration in high-order genome structure at target regions concomitant with the generation of chromatin loops and/or rearrangement of topologically associating domains in a phase-separation-dependent manner. Collectively, these results show that the phase-separated nuclear bodies of nucleoporin fusion proteins can enhance the activation of target genes by promoting the condensation of MLL1/CRM1 and rearrangement of the 3D genome structure.

An in vitro culture platform for studying the effect of collective cell migration on spatial self-organization within induced pluripotent stem cell colonies.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) provide an in vitro system to identify the impact of cell behavior on the earliest stages of cell fate specification during human development. Here, we developed an hiPSC-based model to study the effect of collective cell migration in meso-endodermal lineage segregation and cell fate decisions through the control of space confinement using a detachable ring culture system. RESULTS: The actomyosin organization of cells at the edge of undifferentiated colonies formed in a ring barrier differed from that of the cells in the center of the colony. In addition, even in the absence of exogenous supplements, ectoderm, mesoderm, endoderm, and extraembryonic cells differentiated following the induction of collective cell migration at the colony edge by removing the ring-barrier. However, when collective cell migration was inhibited by blocking E-cadherin function, this fate decision within an hiPSC colony was altered to an ectodermal fate. Furthermore, the induction of collective cell migration at the colony edge using an endodermal induction media enhanced endodermal differentiation efficiency in association with cadherin switching, which is involved in the epithelial-mesenchymal transition. CONCLUSIONS: Our findings suggest that collective cell migration can be an effective way to drive the segregation of mesoderm and endoderm lineages, and cell fate decisions of hiPSCs.

ACAGT-007a, an anti-cancer compound that modulates ERK MAPK signaling, induces nuclear enrichment of phosphorylated ERK in T3M4 pancreatic cancer cells.

The extracellular-signal-regulated-kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as pancreatic cancers. ACAGT-007a (GT-7), an anti-cancer compound, stimulates ERK phosphorylation, thereby inducing growth inhibition and apoptosis in T3M4 pancreatic cancer cells. However, how GT-7 stimulates ERK phosphorylation and induces apoptosis in ERK-active T3M4 cells remains unclear. To look into the mechanism, we performed a spatiotemporal analysis of ERK phosphorylation mediated by GT-7 in T3M4 cells. The immunoblotting showed that GT-7 stimulates ERK phosphorylation within 1 h, which was more remarkable after 2 h. Importantly, apoptosis induction as evaluated by the cleaved Caspase-3 was observed only after 2-h incubation with GT-7. The immunofluorescence staining revealed the enrichment of phosphorylated ERK (phospho-ERK) in the nucleus upon 1-h incubation with GT-7. Fractionation experiments showed that GT-7 increases phospho-ERK levels in the cytoplasm within 1 h, whereas nuclear phospho-ERK accumulation is observed after 2-h incubation with GT-7. MEK inhibition by U0126 significantly diminishes nuclear phospho-ERK distribution and apoptosis induction stimulated by GT-7. Thus, GT-7 may initiate the induction of ERK phosphorylation in the cytoplasm, which leads to phospho-ERK enrichment in the nucleus. This nuclear phospho-ERK accumulation by GT-7 precedes and may underlie apoptosis induction in T3M4.

Recent Advances in Cell Sheet Engineering: From Fabrication to Clinical Translation.

Cell sheet engineering, a scaffold-free tissue fabrication technique, has proven to be an important breakthrough technology in regenerative medicine. Over the past two decades, the field has developed rapidly in terms of investigating fabrication techniques and multipurpose applications in regenerative medicine and biological research. This review highlights the most important achievements in cell sheet engineering to date. We first discuss cell sheet harvesting systems, which have been introduced in temperature-responsive surfaces and other systems to overcome the limitations of conventional cell harvesting methods. In addition, we describe several techniques of cell sheet transfer for preclinical (in vitro and in vivo) and clinical trials. This review also covers cell sheet cryopreservation, which allows short- and long-term storage of cells. Subsequently, we discuss the cell sheet properties of angiogenic cytokines and vasculogenesis. Finally, we discuss updates to various applications, from biological research to clinical translation. We believe that the present review, which shows and compares fundamental technologies and recent advances in cell engineering, can potentially be helpful for new and experienced researchers to promote the further development of tissue engineering in different applications.

Localized Bullous Pemphigoid in a Patient with Acquired Reactive Perforating Collagenosis.

A 61-year-old man presented with 6-month and 5-day histories of multiple, pruritic nodular eruptions on the trunk and extremities and bullous eruptions on the left foot, respectively. The nodular eruptions had been treated with topical corticosteroids without improvement. He had been diagnosed with diabetes mellitus at the age of 42 years and had been suffering from end-stage renal disease for 1 year. Physical examination revealed scattered violet-brown papules and nodules on the trunk and extremities, many of which had central umbilicated necrosis or keratin plugs. Additionally, two tense bullae and five erosions were noted on the dorsal aspect of the left foot. Laboratory tests showed elevated levels of serum anti-bullous pemphigoid (BP)180 antibody. Histopathological findings of a nodule and a bulla were compatible with those of acquired reactive perforating collagenosis (ARPC) and BP, respectively. The papular and nodular lesions were diagnosed as ARPC, while bullous and erosive lesions were diagnosed as localized BP. The present case, together with previously reported cases of coexisting generalized BP and ARPC, suggests that coexistence of BP, regardless of whether generalized or localized, is significantly associated with ARPC.

Novel strategy to improve hepatocyte differentiation stability through synchronized behavior-driven mechanical memory of iPSCs.

Cellular homeostasis is assumed to be regulated by the coordination of dynamic behaviors. Lack of efficient methods for synchronizing large quantities of cells makes studying cell culture strategies for bioprocess development challenging. Here, we demonstrate a novel application of botulinum hemagglutinin (HA), an E-cadherin function-blocking agent, to synchronize behavior-driven mechanical memory in human induced pluripotent stem cell (hiPSC) cultures. Application of HA to hiPSCs resulted in a decrease in actin bundling and disruption of colony formation in a concentration-and time-dependent manner. Interestingly, cytoskeleton rearrangement in cells with prolonged exposure to HA resulted in mechanical memory synchronization with Yes-associated protein, which increased pluripotent cell homogeneity. Synchronized hiPSCs have higher capability to differentiate into functional hepatocytes than unsynchronized hiPSCs, resulting in improved efficiency and robustness of hepatocyte differentiation. Thus, our strategy for cell behavior synchronization before differentiation induction provides an approach against the instability of differentiation of pluripotent cells.

Stable and efficient generation of functional iPSC-derived neural progenitor cell rosettes through regulation of collective cell-cell behavior.

Although the potential of stem cells to differentiate into several cell types has shown promise in regenerative medicine, low differentiation efficiency and poor reproducibility significantly limit their practical application. We developed an effective and robust differentiation strategy for the efficient and robust generation of neural progenitor cell rosettes from induced pluripotent stem cells (iPSCs) incorporating botulinum hemagglutinin (HA). Treatment with HA suppressed the spontaneous differentiation of iPSCs cultured under undirected differentiation conditions, resulting in the preservation of their pluripotency. Moreover, treatment with HA during neural progenitor differentiation combined with dual SMAD inhibition generated a highly homogeneous population of PAX6-and SOX1-expressing neural progenitor cells with 8.4-fold higher yields of neural progenitor cells than untreated control cultures. These neural progenitor cells formed radially organized rosettes surrounding the central lumen. This differentiation method enhanced the generation of functional iPSC-derived neural progenitor cell rosettes throughout the culture vessel, suggesting that the regulation of collective cell-cell behavior using HA plays a morphogenetically important role in rosette formation and maturation. These findings show the significance of HA in the suppression of spontaneous differentiation through spatial homogeneity. The study proposes a novel methodology for the efficient derivation of functional iPSC-derived neural progenitor cell rosettes.

Case report: Severe non-pigmenting fixed drug eruption showing general symptoms caused by chondroitin sulfate sodium.

Non-pigmenting fixed drug eruption (NPFDE) is a subtype of fixed drug eruption (FDE) in which repeated eruptions occur at the same site. Clinically, NPFDE disappears without pigmentation changes; however, it sometimes causes fever or arthralgia. Its histopathological characteristics reportedly include infiltrations of CD8-positive T cells with a paucity of melanocytes as compared to FDE. We present the first case of severe NPFDE exhibiting general symptoms caused by chondroitin sulfate sodium. The patient was a 44-year-old man. Intravenous injection of chondroitin sulfate sodium caused erythema in the affected area. A histopathological examination of the biopsy tissue revealed infiltration of CD3-positive lymphocytes (both CD4-positive and CD8-positive lymphocytes) into the epidermis, minimal liquefaction degeneration in the basal layer of the epidermis, and few dermal melanophages, which may be responsible for non-pigmentation.

Effect of Rho-Associated Kinase Inhibitor on Growth Behaviors of Human Induced Pluripotent Stem Cells in Suspension Culture.

Rho-associated protein kinase (ROCK) inhibitors are used for the survival of single-dissociated human induced pluripotent stem cells (hiPSCs); however, their effects on the growth behaviors of hiPSCs in suspension culture are unexplored. Therefore, we investigated the effect of ROCK inhibitor on growth behaviors of two hiPSC lines (Tic and 1383D2) with different formation of aggregate that attached between single cells in suspension culture. The apparent specific growth rate by long-term exposure to Y-27632, a ROCK inhibitor, was maintained throughout the culture. Long-term exposure to ROCK inhibitor led to an increase in cell division throughout the culture in both lines. Immunofluorescence staining confirmed that hiPSCs forming spherical aggregates showed localization of collagen type I on its periphery. In addition, phosphorylated myosin (pMLC) was localized at the periphery in culture under short-term exposure to ROCK inhibitor, whereas pMLC was not detected at whole the aggregate in culture under long-term exposure. Scanning electron microscopy indicated that long-term exposure to ROCK inhibitor blocked the structural alteration on the surface of cell aggregates. These results indicate that pMLC inhibition by long-term ROCK inhibition leads to enhanced growth abilities of hiPSCs in suspension culture by maintaining the structures of extracellular matrices.

Cell Behavioral Dynamics as a Cue in Optimizing Culture Stabilization in the Bioprocessing of Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) are important for future regenerative medicine therapies. However, in the production of PSCs and derivatives, the control of culture-induced fluctuations in the outcome of cell quality remains challenging. A detailed mechanistic understanding of how PSC behaviors are altered in response to biomechanical microenvironments within a culture is necessary for rational bioprocessing optimization. In this review, we discuss recent insights into the role of cell behavioral and mechanical homeostasis in modulating the states and functions of PSCs during culture processes. We delineate promising ways to manipulate the culture variability through regulating cell behaviors using currently developed tools. Furthermore, we anticipate their potential implementation for designing a culture strategy based on the concept of Waddington's epigenetic landscape that may provide a feasible solution for tuning the culture quality and stability in the bioprocessing space.

Study protocol for periodontal tissue regeneration with a mixture of autologous adipose-derived stem cells and platelet rich plasma: A multicenter, randomized, open-label clinical trial.

Introduction: Adipose-derived stem cells (ASCs) secrete various growth factors to promote wound healing and to regenerate various tissues, such as bone, cartilage, and fat tissue. Subcutaneous adipose tissue is a considerable cell source in clinical practice and can be collected relatively easily and safely under local anesthesia. Moreover, platelet-rich plasma (PRP), a plasma component containing many platelets purified by centrifuging the collected blood, also promotes wound healing. PRP can be easily gelled and is therefore attracting attention as a scaffolding material for transplanted cells. The usefulness of a mixture of ASCs and PRP for periodontal tissue regeneration has been in vitro demonstrated in our previous study. The aim of this study is to present the protocol of translation of tissue regeneration with ASCs and PRP into practical use, evaluating its efficacy. Methods: This study is a multicenter, randomized, open-label comparative clinical trial. Fifteen patients will be randomly assigned to the treatment with mixture of ASCs and PRP or enamel matrix derivate administration into periodontal tissue defects. Increase in height of new alveolar bone in the transplanted area will be evaluated. The evaluation will be performed using dental radiographs after 36 weeks of transplantation. Occurrence of adverse events will be evaluated as secondary outcome. Results: This clinical study was initiated after meeting the regulations to be complied with, including ethical review and regulatory notifications. Conclusions: If effective, this cell therapy using autologous mesenchymal stem cells can represent a useful medical technology for regeneration of periodontal defects.