Expert Revision of Key Elements for Clinical-Grade Production and Qualification of Perinatal Derivatives.

Perinatal derivatives have been proposed as adjunct therapeutic strategies or innovative treatments. Undoubtedly, perinatal derivatives can offer the opportunity and source material to isolate multipotent stem cells, but both maternal- and fetal-derived tissues can be processed and transformed into engineered tissues or advanced biomedical devices, whose potential remains to be fully elucidated. Promising preclinical and clinical results collected so far clearly foresee an escalation of such novel treatments. Market forecasts predict exponential growth in such advanced medicinal products during the next decade, with a pragmatic innovation for medicine into a more advanced biomedical version, enlarging the portfolio for treating a wide range of congenital and acute conditions. However, all these promising and fascinating therapeutic possibilities cannot gain a solid and recognized role in established medical practice without rigid and harmonized manufacturing strategies. The implementation of strategies according to guidelines and directives compiled by Regulatory Agencies, in conformity to (European) Pharmacopoeia and for Good Manufacturing Practice -conforming production of such products, represent critical steps required to translate perinatal technologies into effective therapeutic approaches. During the past 5 years, a panel of European experts and developers, gathered under the umbrella of the COST Sprint Action, supported by the European Cooperation in Science and Technology action, had the opportunity to revise and summarize experience and recommendations for a fruitful and proficient generation of perinatal biomedical products. In order to facilitate the creation and potential commercialization of perinatal bioengineered and advanced pharmaceutical products and technologies, such a collection of data and recommendations is described and discussed here.

Perinatal derivatives: How to best characterize their multimodal functions in vitro. Part C: Inflammation, angiogenesis, and wound healing.

Perinatal derivatives (PnD) are birth-associated tissues, such as placenta, umbilical cord, amniotic and chorionic membrane, and thereof-derived cells as well as secretomes. PnD play an increasing therapeutic role with beneficial effects on the treatment of various diseases. The aim of this review is to elucidate the modes of action of non-hematopoietic PnD on inflammation, angiogenesis and wound healing. We describe the source and type of PnD with a special focus on their effects on inflammation and immune response, on vascular function as well as on cutaneous and oral wound healing, which is a complex process that comprises hemostasis, inflammation, proliferation (including epithelialization, angiogenesis), and remodeling. We further evaluate the different in vitro assays currently used for assessing selected functional and therapeutic PnD properties. This review is a joint effort from the COST SPRINT Action (CA17116) with the intention to promote PnD into the clinics. It is part of a quadrinomial series on functional assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer activities, anti-inflammation, wound healing, angiogenesis, and regeneration.

Perinatal mesenchymal stromal cells of the human decidua restore continence in rats with stress urinary incontinence induced by simulated birth trauma and regulate senescence of fibroblasts from women with stress urinary incontinence.

Stress urinary incontinence (SUI) is a condition that causes the involuntary loss of urine when making small efforts, which seriously affects daily life of people who suffer from it. Women are more affected by this form of incontinence than men, since parity is the main risk factor. Weakening of the pelvic floor tissues is the cause of SUI, although a complete understanding of the cellular and molecular mechanisms of the pathology is still lacking. Reconstructive surgery to strengthen tissue in SUI patients is often associated with complications and/or is ineffective. Mesenchymal stromal cells from the maternal side of the placenta, i.e. the decidua, are proposed here as a therapeutic alternative based on the regenerative potential of mesenchymal cells. The animal model of SUI due to vaginal distention simulating labor has been used, and decidual mesenchymal stromal cell (DMSC) transplantation was effective in preventing a drop in pressure at the leak point in treated animals. Histological analysis of the urethras from DMSC-treated animals after VD showed recovery of the muscle fiber integrity, low or no extracellular matrix (ECM) infiltration and larger elastic fibers near the external urethral sphincter, compared to control animals. Cells isolated from the suburethral connective tissue of SUI patients were characterized as myofibroblasts, based on the expression of several specific genes and proteins, and were shown to achieve premature replicative senescence. Co-culture of SUI myofibroblasts with DMSC via transwell revealed a paracrine interaction between the cells through signals that mediated DMSC migration, SUI myofibroblast proliferation, and modulation of the proinflammatory and ECM-degrading milieu that is characteristic of senescence. In conclusion, DMSC could be an alternative therapeutic option for SUI by counteracting the effects of senescence in damaged pelvic tissue.

[Maximum compressive stress analysis in upper central incisors rehabilitated with fiberglass posts and three types of crowns. A study with the finite element method]. / Análisis de máxima tensión compresiva en incisivos centrales superiores rehabilitados con postes de fibra de vidrio y tres tipos de coronas. Un estudio con método de elementos finitos.

Objective: To evaluate the maximum compressive stress in maxillary central incisors restored with fiberglass posts and three types of crowns by the FEM finite element method. Materials and methods: The study was a virtual, descriptive, and laboratory trial. Three virtual models were made using the SolidWorks 2017 software from upper central incisors rehabilitated with fiberglass posts and a metal-ceramic crown, a monolithic lithium disilicate crown, and a zirconium-ceramic crown. They were then subjected to an oblique occlusal load of 150N with an angulation of 45°, distributed towards the palatal aspect. The stress analysis proceeded by comparing the maximum, minimum, and equivalent von Mises stresses. Results: The maximum compressive stress was found at the cervical level in the vestibular area of each of the crowns. Zirconium-ceramic crown (Design 3) was the one with the highest compressive stress with 73.89 MPa, followed by Lithium Disilicate crown (Design 2) with 63.42 MPa and the metal-ceramic crown (Design 1) with 48.4 MPa. Conclusion: The zirconium-ceramic crown better distributes the stress along the tooth since, due to its rigidity, it absorbs the stresses that are concentrated especially in the cervical area, which could indicate that it is the most appropriate option to rehabilitate endodontically treated teeth.

New Therapeutic Approaches for Allergy: A Review of Cell Therapy and Bio- or Nano-Material-Based Strategies.

Allergy constitutes a major health issue due to its large prevalence. The established therapeutic approaches (allergen avoidance, antihistamines, and corticosteroids) do not address the underlying causes of the pathology, highlighting the need for other long-term treatment options. Antigen-specific immunotherapy enables the long-term control of allergic diseases by promoting immunological tolerance to the allergen. However, efficacious immunotherapies are not available for all possible allergens, and the risk of undesired reactions during therapy remains a concern, especially in patients with severe allergic reactions. In this context, two types of therapeutic strategies appear especially promising for the future in the context of allergy: cell therapy and bio- or nano-material-based therapy. In this review, the main strategies developed this far in these two types of strategies are discussed, with several examples illustrating the different approaches.

Cell therapy for factor V deficiency: An approach based on human decidua mesenchymal stem cells.

Deficiency of factor V is a congenital autosomal recessive coagulopathy associated with mutations in the F5 gene that results in mild-to-severe bleeding episodes. Factor V is a component of the prothrombinase complex responsible for accelerating conversion of prothrombin to thrombin. At the present time there are no therapeutic factor V concentrates available. This study was designed to lay the preliminary foundations for future cell-based therapy for patients with severe factor V deficiency. The study showed that hepatospheres, which produce coagulation factors VIII, IX, and V, synthetize and store intracellular glycogen and express albumin levels up to 8 times higher than those of undifferentiated cells. Factor IX and factor V gene expression increased significantly in hepatospheres as compared to undifferentiated cells, whereas factor VIII gene expression remained constant. The factor V protein was detected in the hepatospheres´ secretome. Considering the enormous potential of mesenchymal stem cells as therapeutic agents, this study proposes a highly reproducible method to induce differentiation of mesenchymal stem cells from human placenta to factor V-producing hepatospheres. This strategy constitutes a preliminary step towards a curative treatment of factor V deficiency through advanced therapies such as cell therapy.

Endostatin Genetically Engineered Placental Mesenchymal Stromal Cells Carrying Doxorubicin-Loaded Mesoporous Silica Nanoparticles for Combined Chemo- and Antiangiogenic Therapy.

Combination therapies constitute a powerful tool for cancer treatment. By combining drugs with different mechanisms of action, the limitations of each individual agent can be overcome, while increasing therapeutic benefit. Here, we propose employing tumor-migrating decidua-derived mesenchymal stromal cells as therapeutic agents combining antiangiogenic therapy and chemotherapy. First, a plasmid encoding the antiangiogenic protein endostatin was transfected into these cells by nucleofection, confirming its expression by ELISA and its biological effect in an ex ovo chick embryo model. Second, doxorubicin-loaded mesoporous silica nanoparticles were introduced into the cells, which would act as vehicles for the drug being released. The effect of the drug was evaluated in a coculture in vitro model with mammary cancer cells. Third, the combination of endostatin transfection and doxorubicin-nanoparticle loading was carried out with the decidua mesenchymal stromal cells. This final cell platform was shown to retain its tumor-migration capacity in vitro, and the combined in vitro therapeutic efficacy was confirmed through a 3D spheroid coculture model using both cancer and endothelial cells. The results presented here show great potential for the development of combination therapies based on genetically-engineered cells that can simultaneously act as cellular vehicles for drug-loaded nanoparticles.

Premature senescence of placental decidua cells as a possible cause of miscarriage produced by mycophenolic acid.

BACKGROUND: Successful pregnancy is supported by a healthy maternal-fetal interface (i.e., the decidual tissues) which holds the conceptus and safeguards it against stressors from the beginning of pregnancy. Any disturbance of this interface can presumably lead to the loss of pregnancy. The use of the immunosuppressive drug mycophenolic acid (MPA) should be discontinued in pregnancy given its abortive and embryotoxic effects. Direct teratogenic effects have been observed in mammalian embryos cultured in MPA, but the underlying mechanisms of abortion by MPA are less understood. METHODS: Decidual stromal cells isolated from human placentas are cultured in the presence of clinically relevant doses of MPA. Data regarding the effects of MPA on the proliferation and viability of decidua cultures are first analysed and then, molecular pathways contributing to these effects are unravelled. RESULTS: MPA treatment of decidual stromal cells results in loss of proliferation capacity and a decrease in the viability of decidua cultures. The molecular pathways involved in the effects of MPA on decidual stromal cells are a reduction in pre-rRNA synthesis and subsequent disruption of the nucleolus. The nucleolar stress stabilizes p53, which in turn, leads to a p21-mediated cell cycle arrest in late S and G2 phases, preventing the progression of the decidua cells into the mitosis. Furthermore, MPA does not induce apoptosis but activate mechanisms of autophagy and senescence in decidual stromal cells. CONCLUSION: The irreversible growth arrest of decidua cells, whose role in the maintenance of the pregnancy microenvironment is known, may be one cause of miscarriage in MPA treated pregnant women.

Current Status and Future Prospects of Perinatal Stem Cells.

The placenta is a temporary organ that is discarded after birth and is one of the most promising sources of various cells and tissues for use in regenerative medicine and tissue engineering, both in experimental and clinical settings. The placenta has unique, intrinsic features because it plays many roles during gestation: it is formed by cells from two individuals (mother and fetus), contributes to the development and growth of an allogeneic fetus, and has two independent and interacting circulatory systems. Different stem and progenitor cell types can be isolated from the different perinatal tissues making them particularly interesting candidates for use in cell therapy and regenerative medicine. The primary source of perinatal stem cells is cord blood. Cord blood has been a well-known source of hematopoietic stem/progenitor cells since 1974. Biobanked cord blood has been used to treat different hematological and immunological disorders for over 30 years. Other perinatal tissues that are routinely discarded as medical waste contain non-hematopoietic cells with potential therapeutic value. Indeed, in advanced perinatal cell therapy trials, mesenchymal stromal cells are the most commonly used. Here, we review one by one the different perinatal tissues and the different perinatal stem cells isolated with their phenotypical characteristics and the preclinical uses of these cells in numerous pathologies. An overview of clinical applications of perinatal derived cells is also described with special emphasis on the clinical trials being carried out to treat COVID19 pneumonia. Furthermore, we describe the use of new technologies in the field of perinatal stem cells and the future directions and challenges of this fascinating and rapidly progressing field of perinatal cells and regenerative medicine.

Leachate effects of pelagic Sargassum spp. on larval swimming behavior of the coral Acropora palmata.

An emerging disturbance for Caribbean reefs is the massive arrival of pelagic Sargassum, which deteriorates water quality due to the production of leachates. The highest arrivals of Sargassum took place when broadcasting corals spawned. We experimentally determined the effect of Sargassum leachates on swimming behavior of Acropora palmata larvae through five treatments (control, stain (simulating 100% leachate color), and 25%, 50% and 100% Sargassum leachate concentrations) during 30 min (10 min of videos and 20 min of post-observations). In the videos, larvae with leachates reduced swimming speed, were positively geotactic, the percentage of individuals that swam in a spiral pattern increased, and most behavioral displacements occurred at lower frequencies than larvae without leachates. Moreover, symptomatic spiral behavior was higher in the presence of leachates, suggesting that this behavior may be an effect of pollution. During post-observations, most larvae with leachates were motionless. This is the first time that Sargassum leachates have been documented modifying larval swimming behavior, which may reduce larval dispersion and genetic diversity. We suggest that a future evaluation of the effects of leachates at lower concentrations and over longer periods of exposure is needed. The resilience of corals may be compromised if Sargassum arrivals become frequent events.

Cell-Based Nanoparticles Delivery Systems for Targeted Cancer Therapy: Lessons from Anti-Angiogenesis Treatments.

The main strategy of cancer treatment has focused on attacking the tumor cells. Some cancers initially responsive to chemotherapy become treatment-resistant. Another strategy is to block the formation of tumor vessels. However, tumors also become resistant to anti-angiogenic treatments, mostly due to other cells and factors present in the tumor microenvironment, and hypoxia in the central part of the tumor. The need for new cancer therapies is significant. The use of nanoparticle-based therapy will improve therapeutic efficacy and targeting, while reducing toxicity. However, due to inefficient accumulation in tumor sites, clearance by reticuloendothelial organs and toxicity, internalization or conjugation of drug-loaded nanoparticles (NPs) into mesenchymal stem cells (MSCs) can increase efficacy by actively delivering them into the tumor microenvironment. Nanoengineering MSCs with drug-loaded NPs can increase the drug payload delivered to tumor sites due to the migratory and homing abilities of MSCs. However, MSCs have some disadvantages, and exosomes and membranes from different cell types can be used to transport drug-loaded NPs actively to tumors. This review gives an overview of different cancer approaches, with a focus on hypoxia and the emergence of NPs as drug-delivery systems and MSCs as cellular vehicles for targeted delivery due to their tumor-homing potential.

Suicide-gene transfection of tumor-tropic placental stem cells employing ultrasound-responsive nanoparticles.

A Trojan-horse strategy for cancer therapy employing tumor-tropic mesenchymal stem cells transfected with a non-viral nanovector is here presented. In this sense, ultrasound-responsive mesoporous silica nanoparticles were coated with a polycation (using two different molecular weights), providing them with gene transfection capabilities that were evaluated using two different plasmids. First, the expression of Green Fluorescent Protein was analyzed in Decidua-derived Mesenchymal Stem Cells after incubation with the silica nanoparticles. The most successful nanoparticle was then employed to induce the expression of two suicide genes: cytosine deaminase and uracil phosphoribosyl transferase, which allow the cells to convert a non-toxic pro-drug (5-fluorocytosine) into a toxic drug (5-Fluorouridine monophosphate). The effect of the production of the toxic final product was also evaluated in a cancer cell line (NMU cells) co-cultured with the transfected vehicle cells, Decidua-derived Mesenchymal Stem Cells. STATEMENT OF SIGNIFICANCE: Cell-mediated cancer therapy has recently attracted great interest. Tumor-homing cells can exert anticancer effects through innate capacities, via transfection with a therapeutic gene or acting as vehicles of therapeutic nanoparticles. In this work, an ultrasound-responsive mesoporous silica nanoparticle (capable of carrying an anticancer drug) is engineered to act as a non-viral transfection agent for tumor-tropic human placental mesenchymal stem cells. The successful transfection of the vehicle cells is evaluated employing different expression plasmids. After transfection with two suicide genes, the vehicle cells are capable of converting a non-toxic pro-drug into a highly toxic molecule, which can also kill surrounding cancer cells in an in vitro co-culture model. This work opens the gate for a plethora of strategies in which both genes and drug-loaded nanoparticles can be transported towards tumor tissues by easily available human mesenchymal stem cells.

Searching for a Cell-Based Therapeutic Tool for Haemophilia A within the Embryonic/Foetal Liver and the Aorta-Gonads-Mesonephros Region.

The development of new strategies based on cell therapy approaches to correct haemophilia A (HA) requires further insights into new cell populations capable of producing coagulation factor VIII (FVIII) and presenting stable engraftment potential. The major producers of FVIII in the adult are liver sinusoidal endothelial cells (LSECs) and in a lesser degree bone marrow-derived cells, both of which have been shown to ameliorate the bleeding phenotype in adult HA mice after transplantation. We have previously shown that cells from the foetal liver (FL) and the aorta-gonads-mesonephros (AGM) haematopoietic locations possess higher LSEC engraftment potential in newborn mice compared with adult-derived LSECs, constituting likely therapeutic targets for the treatment of HA in neonates. However, less is known about the production of FVIII in embryonic locations. Quantitative polymerase chain reaction and Western blot analysis were performed to assess the relative level of FVIII production in different embryonic tissues and at various developmental stages, identifying the FL and AGM region from day 12 (E12) as prominent sources of FVIII. Furthermore, FL-derived VE-cad+CD45-Lyve1+/- endothelial/endothelial progenitor cells, presenting vascular engraftment potential, produced high levels of F8 ribonucleic acid compared with CD45+ blood progenitors or Dlk1+ hepatoblasts. In addition, we show that the E11 AGM explant cultures expanded cells with LSEC repopulation activity, instrumental to further understand signals for in vitro generation of LSECs. Taking into account the capacity for FVIII expression, culture expansion and newborn engraftment potential, these results support the use of cells with foetal characteristics for correction of FVIII deficiency in young individuals.

Vectorization of ultrasound-responsive nanoparticles in placental mesenchymal stem cells for cancer therapy.

A new platform constituted by engineered responsive nanoparticles transported by human mesenchymal stem cells is here presented as a proof of concept. Ultrasound-responsive mesoporous silica nanoparticles are coated with polyethylenimine to favor their effective uptake by decidua-derived mesenchymal stem cells. The responsive-release ability of the designed nanoparticles is confirmed, both in vial and in vivo. In addition, this capability is maintained inside the cells used as carriers. The migration capacity of the nanoparticle-cell platform towards mammary tumors is assessed in vitro. The efficacy of this platform for anticancer therapy is shown against mammary tumor cells by inducing the release of doxorubicin only when the cell vehicles are exposed to ultrasound.

Restrained Th17 response and myeloid cell infiltration into the central nervous system by human decidua-derived mesenchymal stem cells during experimental autoimmune encephalomyelitis.

BACKGROUND: Multiple sclerosis is a widespread inflammatory demyelinating disease. Several immunomodulatory therapies are available, including interferon-ß, glatiramer acetate, natalizumab, fingolimod, and mitoxantrone. Although useful to delay disease progression, they do not provide a definitive cure and are associated with some undesirable side-effects. Accordingly, the search for new therapeutic methods constitutes an active investigation field. The use of mesenchymal stem cells (MSCs) to modify the disease course is currently the subject of intense interest. Decidua-derived MSCs (DMSCs) are a cell population obtained from human placental extraembryonic membranes able to differentiate into the three germ layers. This study explores the therapeutic potential of DMSCs. METHODS: We used the experimental autoimmune encephalomyelitis (EAE) animal model to evaluate the effect of DMSCs on clinical signs of the disease and on the presence of inflammatory infiltrates in the central nervous system. We also compared the inflammatory profile of spleen T cells from DMSC-treated mice with that of EAE control animals, and the influence of DMSCs on the in vitro definition of the Th17 phenotype. Furthermore, we analyzed the effects on the presence of some critical cell types in central nervous system infiltrates. RESULTS: Preventive intraperitoneal injection of DMSCs resulted in a significant delay of external signs of EAE. In addition, treatment of animals already presenting with moderate symptoms resulted in mild EAE with reduced disease scores. Besides decreased inflammatory infiltration, diminished percentages of CD4(+)IL17(+), CD11b(+)Ly6G(+) and CD11b(+)Ly6C(+) cells were found in infiltrates of treated animals. Early immune response was mitigated, with spleen cells of DMSC-treated mice displaying low proliferative response to antigen, decreased production of interleukin (IL)-17, and increased production of the anti-inflammatory cytokines IL-4 and IL-10. Moreover, lower RORγT and higher GATA-3 expression levels were detected in DMSC-treated mice. DMSCs also showed a detrimental influence on the in vitro definition of the Th17 phenotype. CONCLUSIONS: DMSCs modulated the clinical course of EAE, modified the frequency and cell composition of the central nervous system infiltrates during the disease, and mediated an impairment of Th17 phenotype establishment in favor of the Th2 subtype. These results suggest that DMSCs might provide a new cell-based therapy for the control of multiple sclerosis.

Decidua-derived mesenchymal stem cells as carriers of mesoporous silica nanoparticles. In vitro and in vivo evaluation on mammary tumors.

The potential use of human Decidua-derived mesenchymal stem cells (DMSCs) as a platform to carry mesoporous silica nanoparticles in cancer therapy has been investigated. Two types of nanoparticles were evaluated. The nanoparticles showed negligible toxicity to the cells, a fast uptake and a long retention inside them. Nanoparticle location in the cell was studied by colocalization with the lysosomes. Moreover, the in vitro and in vivo migration of DMSCs towards tumors was not modified by the evaluated nanoparticles. Finally, DMSCs transporting doxorubicin-loaded nanoparticles were capable of inducing cancer cell death in vitro. STATEMENT OF SIGNIFICANCE: The use of nanotechnology for anticancer drug delivery has recently attracted great interest. Nanoparticles such as mesoporous silica nanoparticles (MSNs) can reach tumors, either by passive targeting, through the enhanced permeability and retention (EPR) effect, or active targeting, through the functionalization of nanoparticle surface. However, nanotechnology has not yet achieved the expected results in improving drug targeting, highlighting the need for a better localization of the nanoparticles in the tumors. Human mesenchymal stem cells from the decidua of the human placenta (DMSCs) have been observed to migrate towards tumors in a preclinical model of breast cancer. Moreover, they have been shown to inhibit growth of primary tumors and development of new tumors. In this work, combining MSNs and DMSCs, we have studied for the first time whether placental stem cells could be employed as a platform to load nanoparticles and carry them towards tumors for future anticancer therapies.

Stem cell therapy in inflammatory bowel disease: A promising therapeutic strategy?

Inflammatory bowel diseases are inflammatory, chronic and progressive diseases of the intestinal tract for which no curative treatment is available. Research in other fields with stem cells of different sources and with immunoregulatory cells (regulatory T-lymphocytes and dendritic T-cells) opens up new expectations for their use in these diseases. The goal for stem cell-based therapy is to provide a permanent cure. To achieve this, it will be necessary to obtain a cellular product, original or genetically modified, that has a high migration capacity and homes into the intestine, has high survival after transplantation, regulates the immune reaction while not being visible to the patient's immune system, and repairs the injured tissue.

Human decidua-derived mesenchymal stem cells differentiate into functional alveolar type II-like cells that synthesize and secrete pulmonary surfactant complexes.

Lung alveolar type II (ATII) cells are specialized in the synthesis and secretion of pulmonary surfactant, a lipid-protein complex that reduces surface tension to minimize the work of breathing. Surfactant synthesis, assembly and secretion are closely regulated and its impairment is associated with severe respiratory disorders. At present, well-established ATII cell culture models are not available. In this work, Decidua-derived Mesenchymal Stem Cells (DMSCs) have been differentiated into Alveolar Type II- Like Cells (ATII-LCs), which display membranous cytoplasmic organelles resembling lamellar bodies, the organelles involved in surfactant storage and secretion by native ATII cells, and accumulate disaturated phospholipid species, a surfactant hallmark. Expression of characteristic ATII cells markers was demonstrated in ATII-LCs at gene and protein level. Mimicking the response of ATII cells to secretagogues, ATII-LCs were able to exocytose lipid-rich assemblies, which displayed highly surface active capabilities, including faster interfacial adsorption kinetics than standard native surfactant, even in the presence of inhibitory agents. ATII-LCs could constitute a highly useful ex vivo model for the study of surfactant biogenesis and the mechanisms involved in protein processing and lipid trafficking, as well as the packing and storage of surfactant complexes.

Human decidua-derived mesenchymal stromal cells differentiate into hepatic-like cells and form functional three-dimensional structures.

BACKGROUND AIMS: Previously, we have shown that human decidua-derived mesenchymal stromal cells (DMSC) are mesenchymal stromal cells (MSC) with a clonal differentiation capacity for the three embryonic layers. The endodermal capacity of DMSC was revealed by differentiation into pulmonary cells. In this study, we examined the hepatic differentiation of DMSC. METHODS: DMSC were cultured in hepatic differentiation media or co-cultured with murine liver homogenate and analyzed with phenotypic, molecular and functional tests. RESULTS AND CONCLUSIONS: DMSC in hepatic differentiation media changed their fibroblast morphology to a hepatocyte-like morphology and later formed a 3-dimensional (3-D) structure or hepatosphere. Moreover, the hepatocyte-like cells and the hepatospheres expressed liver-specific markers such as synthesis of albumin (ALB), hepatocyte growth factor receptor (HGFR), α-fetoprotein (AFP) and cytokeratin-18 (CK-18), and exhibited hepatic functions including glycogen storage capacity and indocyanine green (ICG) uptake/secretion. Human DMSC co-cultured with murine liver tissue homogenate in a non-contact in vitro system showed hepatic differentiation, as evidenced by expression of AFP and ALB genes. The switch in the expression of these two genes resembled liver development. Indeed, the decrease in AFP and increase in ALB expression throughout the co-culture were consistent with the expression pattern observed during normal liver organogenesis in the embryo. Interestingly, AFP and ALB expression was significantly higher when DMSC were co-cultured with injured liver tissue, indicating that DMSC respond differently under normal and pathologic micro-environmental conditions. In conclusion, DMSC-derived hepatospheres and DMSC co-cultured with liver homogenate could be suitable in vitro models for toxicologic, developmental and pre-clinical hepatic regeneration studies.