• Adipocytes
• Adipose-derived stem cells
• Mesenchymal stem cells
• Regenerative medicine

• Humans
• Regenerative Medicine/methods
• Cell Differentiation/physiology
• Adipose Tissue/cytology
• Mesenchymal Stem Cells/cytology
• Mesenchymal Stem Cells/physiology
• Animals
• Stem Cells/cytology
• Tissue Engineering/methods

• cell culture media
• cell therapy manufacturing
• human mesenchymal stromal cells
• immunomodulation
• multipotentiality
• xeno-free

• Humans
• Mesenchymal Stem Cells/cytology
• Mesenchymal Stem Cells/immunology
• Mesenchymal Stem Cells/drug effects
• Mesenchymal Stem Cells/metabolism
• Cell Differentiation/drug effects
• Immunomodulation/drug effects
• Cell Proliferation/drug effects
• Cells, Cultured
• Osteogenesis/drug effects
• Culture Media/pharmacology
• Leukocytes, Mononuclear/cytology
• Chondrogenesis/drug effects
• Immunophenotyping
• Coculture Techniques
• Adipogenesis/drug effects
• Cell Culture Techniques/methods

• Angiogenesis
• Myocardial infarction
• Myogenesis
• Platelet lysate
• Stem cell

• Animals
• Myocardial Infarction/therapy
• Myocardial Infarction/metabolism
• Myocardial Infarction/physiopathology
• Mesenchymal Stem Cells/metabolism
• Mesenchymal Stem Cells/cytology
• Blood Platelets/metabolism
• Male
• Humans
• Mesenchymal Stem Cell Transplantation/methods
• Rats
• Rats, Wistar
• Disease Models, Animal
• Cell Extracts/pharmacology

• IR.KMU.REC. 98000777 Vice-Chancellor for Research and Technology at Kerman University of Medical Sciences, Kerman, Iran
• Grant No. IR.RUMS.REC. 98193 Rafsanjan University of Medical Sciences.

• 5-Azacytidine
• Mesenchymal stem cells
• cardiomyogenic differentiation
• growth factors

• bone regeneration
• calcium phosphates
• dental implants
• safety
• sinus floor augmentation
• stromal vascular fraction

• Humans
• Bone Regeneration
• Bone Substitutes/adverse effects
• Calcium Phosphates/adverse effects
• Ceramics
• Dental Implants
• Prospective Studies
• Sinus Floor Augmentation/methods
• Stromal Vascular Fraction
• Clinical Trials, Phase I as Topic
• Follow-Up Studies

• Health Research
• Health-Holland
• ZonMw

• Cell expansion
• Cell therapy
• Cytokine-induced killer cells (CIK)
• Good Manufacturing Practice (GMP)

• Animals
• Humans
• Leukocytes, Mononuclear
• Cell Culture Techniques
• Cell Cycle
• Cell Proliferation
• Cytokines

• The Iranian Ministry of Health and Medical Education

• cell proliferation
• in vitro studies
• mesenchymal stem/stromal cells
• neuronal differentiation
• serum-free media

• Animals
• Cell Culture Techniques/methods
• Cell Differentiation/physiology
• Cell Proliferation/physiology
• Cells, Cultured
• Culture Media/metabolism
• Culture Media, Serum-Free
• Mesenchymal Stem Cells/metabolism

• Cardiac regenerative therapy
• Cardiomyocytes
• Cell delivery
• Differentiation
• Mesenchymal stem cells

Mesenchymal stem cells (MSCs) are of great interest for their use in cell-based therapies due to their multipotent differentiation and immunomodulatory capacities. In consequence of limited numbers following their isolation from the donor tissue, MSCs require extensive expansion performed in traditional 2D cell culture setups to reach adequate amounts for therapeutic use. However, prolonged culture of MSCs in vitro has been shown to decrease their differentiation potential and alter their immunomodulatory properties. For that reason, preservation of these physiological characteristics of MSCs throughout their in vitro culture is essential for improving the efficiency of therapeutic and in vitro modeling applications. With this objective in mind, many studies already investigated certain parameters for enhancing current standard MSC culture protocols with regard to the effects of specific culture media components or culture conditions. Although there is a lot of diversity in the final therapeutic uses of the cells, the primary stage of standard isolation and expansion is imperative. Therefore, we want to review on approaches for optimizing standard MSC culture protocols during this essential primary step of in vitro expansion. The reviewed studies investigate and suggest improvements focused on culture media components (amino acids, ascorbic acid, glucose level, growth factors, lipids, platelet lysate, trace elements, serum, and xenogeneic components) as well as culture conditions and processes (hypoxia, cell seeding, and dissociation during passaging), in order to preserve the MSC phenotype and functionality during the primary phase of in vitro culture.

• mesenchymal stem cells
• optimization cultivation
• physiologic cultivation

Congenital abnormalities, cancers as well as injuries can cause irreversible damage to the urinary tract, which eventually requires tissue reconstruction. Smooth muscle cells, endothelial cells, and urothelial cells are the major cell types required for the reconstruction of lower urinary tract. Adult stem cells represent an accessible source of unlimited repertoire of untransformed cells.

Fetal bovine serum (FBS) is the most vital supplement in the culture media used for cellular proliferation and differentiation. However, due to the increasing interest in manufacturing xeno-free stem cell-based cellular products, optimizing the composition of the culture media and the serum-type used is of paramount importance. In this study, the effects of FBS and pooled human platelet (pHPL) lysate were assessed on the capacity of human adipose-derived stem cells (ADSCs) to differentiate into urothelial-like cells. Also, we aimed to compare the ability of both conditioned media (CM) and unconditioned urothelial cell media (UCM) to induce urothelial differentiation of ADCS in vitro.

ADSCs were isolated from human lipoaspirates and characterized by flow cytometry for their ability to express the most common mesenchymal stem cell (MSCs) markers. The differentiation potential was also assessed by differentiating them into osteogenic and adipogenic cell lineages. To evaluate the capacity of ADSCs to differentiate towards the urothelial-like lineage, cells were cultured with either CM or UCM, supplemented with either 5% pHPL, 2.5% pHPL or 10% FBS. After 14 days of induction, cells were utilized for gene expression and immunofluorescence analysis.

ADSCs cultured in CM and supplemented with FBS exhibited the highest upregulation levels of the urothelial cell markers; cytokeratin-18 (CK-18), cytokeratin-19 (CK-19), and Uroplakin-2 (UPK-2), with a 6.7, 4.2- and a 2-folds increase in gene expression, respectively. Meanwhile, the use of CM supplemented with either 5% pHPL or 2.5% pHPL, and UCM supplemented with either 5% pHPL or 2.5% pHPL showed low expression levels of CK-18 and CK-19 and no upregulation of UPK-2 level was observed. In contrast, the use of UCM with FBS has increased the levels of CK-18 and CK-19, however to a lesser extent compared to CM. At the cellular level, CK-18 and UPK-2 were only detected in CM/FBS supplemented group. Growth factor analysis revealed an increase in the expression levels of EGF, VEGF and PDGF in all of the differentiated groups.

Efficient ADSCs urothelial differentiation is dependent on the use of conditioned media. The presence of high concentrations of proliferation-inducing growth factors present in the pHPL reduces the efficiency of ADSCs differentiation towards the urothelial lineage. Additionally, the increase in EGF, VEGF and PDGF during the differentiation implicates them in the mechanism of urothelial cell differentiation.

• Adipose stem cells
• Cellular differentiation
• Pooled human platelet lysate
• Urothelial cells

Cerebral ischemia-reperfusion (I/R) injury, the most common form of stroke, has high mortality and often brings persistent and serious brain dysfunction among survivors. Administration of adipose-derived mesenchymal stem cells (ASCs) has been suggested to alleviate the I/R brain injury, but the mechanism remains uncharacterized. Here, we aimed at investigating the mechanism of ASCs and their extracellular vesicles (EVs) in the repair of or protection from I/R injury.

We established the middle cerebral artery occlusion (MCAO) model and oxygen-glucose deprivation/reperfusion (OGD/RP) neuron model. ASCs or ASC-derived EVs (ASC-EVs) were co-cultured with neurons. RT-qPCR and Western blot analyses determined microRNA (miRNA)-22-3p, BMP2, BMF, and KDM6B expression in neurons upon treatment with ASC-EVs. Bioinformatics analysis predicted the binding between miR-22-3p and KDM6B. Using gain- and loss-of-function methods, we tested the impact of these molecules on I/R injury in vivo and in vitro.

Treatment with ASCs and ASC-derived EVs significantly alleviated the I/R brain injury in vivo, elevated neuron viability in vitro, and decreased apoptosis. Interestingly, miR-22-3p was upregulated in ASC-EVs, and treatment with EV-miR-22-3p inhibitor led to increased apoptosis and decreased neuronal. Of note, miR-22-3p bound to and inhibited KDM6B, as demonstrated by dual-luciferase reporter gene assay and Western blot assay. Overexpression of KDM6B enhanced apoptosis of neurons in the OGD/RP model, and KDM6B bound to BMB2 and promoted its expression by binding to BMP2. Silencing of BMF reduced infarct volume and apoptosis in the stroke model.

Results support a conclusion that ASC-EV-derived miR-22-3p could alleviate brain ischemic injury by inhibiting KDM6B-mediated effects on the BMP2/BMF axis. These findings compelling indicate a novel treatment strategy for cerebral ischemic injury.

• Adipose-derived mesenchymal stem cells
• BMF
• BMP2
• Extracellular vesicles
• Ischemia-reperfusion
• KDM6B
• microRNA-22-3p

• adipose-derived mesenchymal stem cells
• bone tissue engineering
• hyperoxia
• hypoxia
• normoxia
• vascularization

• adipose-derived stem cell
• angiogenesis
• cell sheet
• extracellular matrix
• fetal bovine serum
• human platelet lysate

Human amniotic fluid mesenchymal stem cells (hAF-MSCs) have been shown to be effective in the treatment of many diseases. Platelet lysate (PL) contains multiple growth and differentiation factors; therefore, it can be used as a differentiation inducer. In this study, we attempted to evaluate the efficiency of human platelet lysate (hPL) on cell viability and the effects on cardiomyogenic differentiation of hAF-MSCs. When treating the cells with hPL, the result showed an increase in cell viability. Expressions of cardiomyogenic specific genes, including GATA4, cTnT, Cx43 and Nkx2.5, were higher in the combined treatment groups of 5-azacytidine (5-aza) and hPL than the expressions of cardiomyogenic specific genes in the control group and in the 5-aza treatment group. In terms of the results of immunofluorescence and immunoenzymatic staining, the highest expressions of cardiomyogenic specific proteins were revealed in combined treatment groups. It can be summarized that hPL may be an effective supporting cardiomyogenic supplementary factor for cardiomyogenic differentiation in hAF-MSCs.

• 5-azacytidine
• Biomedical engineering
• Cardiomyocyte-like cells
• Cardiomyogenic differentiation
• Cell biology
• Human amniotic fluid mesenchymal stem cells
• Human platelet lysate
• Molecular biology
• Regenerative medicine
• Stem cell research

• Adipose derived stem cells
• Cell therapy
• Foetal bovine serum
• Human platelet lysate
• Mesenchymal stem cells

Adipose‐derived stem cells (ASC) are said to have a pivotal role in wound healing. Specifically, ASC‐secreted extracellular vesicles (EV) carry diverse cargos such as microRNAs (miRNAs) to participate in the ASC‐based therapies. Considering its effects, we aimed to investigate the role of ASC‐EVs in the cutaneous wound healing accompanied with the study on the specific cargo‐medicated effects on wound healing. Two full‐thickness excisional skin wounds were created on mouse dorsum, and wound healing was recorded at the indicated time points followed by histological analysis and immunofluorescence staining for CD31 and α‐SMA. Human skin fibroblasts (HSFs) and human microvascular endothelial cells (HMECs) were co‐cultured with EVs isolated from ASC (ASC‐EVs), respectively, followed by the evaluation of their viability and mobility using CCK‐8, scratch test and transwell migration assays. Matrigel‐based angiogenesis assays were performed to evaluate vessel‐like tube formation by HMECs in vitro. ASC‐EVs accelerated the healing of full‐thickness skin wounds, increased re‐epithelialization and reduced scar thickness whilst enhanced collagen synthesis and angiogenesis in murine models. However, miR‐486‐5p antagomir abrogated the ASC‐EVs‐induced effects. Intriguingly, miR‐486‐5p was found to be highly enriched in ASC‐EVs, exhibiting an increase in viability and mobility of HSFs and HMECs and enhanced the angiogenic activities of HMECs. Notably, we also demonstrated that ASC‐EVs‐secreted miR‐486‐5p achieved the aforesaid effects through its target gene Sp5. Hence, our results suggest that miR‐486‐5p released by ASC‐EVs could be a critical mediator to develop an ASC‐based therapeutic strategy for wound healing.

• adipose-derived stem cells
• angiogenesis
• cutaneous wound
• extracellular vesicles
• microRNA-486-5p

• Bio-product
• Clinical applications
• Platelet Rich Plasma (PRP)
• Platelets
• Regenerative medicine

• Growth factor delivery
• Microgels
• Platelet lysate

• Blood Platelets/cytology
• Drug Carriers/chemistry
• Drug Liberation
• Humans
• Hyaluronic Acid/chemistry
• Microgels/chemistry
• Tissue Engineering

• SDF-1
• acute myocardial infarction
• in vivo
• targeted microbubbles
• ultrasound molecular imaging

• adipogenesis
• adipose-derived stromal/stem cells
• chondrogenesis
• colony forming unit-fibroblast
• fetal bovine serum
• human platelet lysate
• mesenchymal stem cell
• osteogenesis
• regenerative medicine

Adipose stromal/stem cells (ASCs) are an ideal cell type for regenerative medicine applications, as they can easily be harvested from adipose tissue in large quantities. ASCs have excellent proliferation, differentiation, and immunoregulatory capacities that have been demonstrated in numerous studies. Great interest and investment have been placed in efforts to exploit the allogeneic use and immunomodulatory and anti-inflammatory effects of ASCs. However, bridging the gap between in vitro and in vivo studies and moving into clinical practice remain a challenge. For the clinical translation of ASCs, several issues must be considered, including how to characterise such a heterogenic cell population and how to ensure their safety and efficacy. This review explores the different phases of in vitro and preclinical ASC characterisation and describes the development of appropriate potency assays. In addition, good manufacturing practice requirements are discussed, and cell-based medicinal products holding marketing authorisation in the European Union are reviewed. Moreover, the current status of clinical trials applying ASCs and the patent landscape in the field of ASC research are presented. Overall, this review highlights the applicability of ASCs for clinical cell therapies and discusses their potential.

Human amniotic fluid (hAF) mesenchymal stem cells (MSCs) are commonly cultured in medium containing FBS. However, there are concerns about using animal serum in therapeutic applications due to the potential for immunogenic reactions and the risk of transmission of pathogens. For safety reasons, human platelet lysate (hPL) has been suggested as a replacement for FBS because it appears to be a natural source of growth factors. In this present study, it was investigated whether FBS could be substituted with hPL in hAF-MSCs culture without affecting their properties. Pooled hPL was generated by the freeze-thaw method. The concentration of hPL was selected after evaluation by MTT assay. The hAF-MSCs were cultured in FBS- or hPL-supplemented conditions and shared a fibroblast-like morphology. Cell proliferation assays showed that the growth characteristic of hAF-MSCs cultured in 10% hPL-supplemented media was similar to those cultured in 10% FBS-supplemented media. The expression of MSC markers did not differ between the cells cultured in the different conditions. The endothelial differentiation potential was also investigated. Reverse transcription-quantitative (RT-q)PCR revealed that induced cells supplemented with hPL showed an increase level of endothelial specific gene expression compared to the FBS-supplemented cells. Immunofluorescence analysis showed specific protein localization in both induced cell groups. Additionally, induced cells supplemented with hPL had the potential to form networks on Matrigel. This present study indicated that hPL could be used to culture and enhance the endothelial differentiation potential of hAF-MSCs.

• Wharton's jelly cells
• extracellular matrix proteins
• immunogenicity
• immunomodulatory
• mesenchymal stromal cells
• serum

• Adipose-derived stromal cells
• bioreactor
• clinical application
• closed cultivation systems
• cryoprecipitate
• human platelet lysate
• mesenchymal stromal cells
• stem cells

• Additive solution
• adipose-derived stromal cells
• expired buffy coats
• foetal bovine serum
• pooled human platelet lysate

Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs) by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL-) cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells.

Tissue engineering technology offers an appealing approach for tissue reconstruction of the urothelium. Adipose-derived mesenchymal stem cells (ADSCs) represent an abundant source for tissue engineering applications. However, ASCs primarily possess mesoderm lineage differentiation potential. It is difficult to induce differentiation of ASCs towards urothelial cells that are derived from the endoderm, although a recent findings have reported that a conditioned medium may drive ADSCs towards differentiation into the urothelium phenotype. In the present study, human ADSCs were isolated from abdominal adipose tissues and incubated in this conditioned medium for indicated time periods. Western blotting showed that protein expression levels of urothelial specific marks, including CK7, CK20 and UPIII, were increased after seven days' incubation, but immunofluorescence microscopy determined that cells with CK7 and UPIII staining were scarce, which suggested a low-efficiency for the differentiation. Prolonging the incubation time did not further increase CK20 and UPIII expression. Furthermore, miR-33a expression was increased with ADSC differentiation. Using synthetic miRNAs to mimic or inhibit the action of miR-33a revealed that miR-33a hinders the differentiation of ADSCs towards urothelial cells. Furthermore, luciferase reporter assay confirmed that β-catenin and transforming growth factor-β receptor (TGFR) are targets of miR-33a. Inhibition of miR-33a expression increased β-catenin and TGFR expression and improved the efficiency of ADSCs towards differentiation into the urothelium phenotype. The present novel finding suggests that miR-33 may be an important target in tissue engineering and regenerative medicine for urothelium repair.

• cell isolation
• enzymatic isolation
• explant culture
• extracellular vesicles
• human mesenchymal stem cells

An X-linked condition characterized by the combination of hypomyelinating leukodystrophy and spondylometaphyseal dysplasia (H-SMD) has been observed in only four families, with linkage to Xq25-27, and recent genetic characterization in two families with a common AIFM1 mutation. In our study, 12 patients (6 families) with H-SMD were identified and underwent comprehensive assessment accompanied by whole-exome sequencing (WES). Pedigree analysis in all families was consistent with X-linked recessive inheritance. Presentation typically occurred between 12 and 36 months. In addition to the two disease-defining features of spondylometaphyseal dysplasia and hypomyelination on MRI, common clinical signs and symptoms included motor deterioration, spasticity, tremor, ataxia, dysarthria, cognitive defects, pulmonary hypertension, nystagmus, and vision loss due to retinopathy. The course of the disease was slowly progressive. All patients had maternally inherited or de novo mutations in or near exon 7 of AIFM1, within a region of 70 bp, including synonymous and intronic changes. AIFM1 mutations have previously been associated with neurologic presentations as varied as intellectual disability, hearing loss, neuropathy, and striatal necrosis, while AIFM1 mutations in this small region present with a distinct phenotype implicating bone. Analysis of cell lines derived from four patients identified significant reductions in AIFM1 mRNA and protein levels in osteoblasts. We hypothesize that AIFM1 functions in bone metabolism and myelination and is responsible for the unique phenotype in this condition.

• AIFM1 gene
• Hypomyelination
• Mitochondrial leukodystrophy
• Myelin
• Spondylometaphyseal dysplasia
• Whole exome sequencing (WES)

• cell therapy
• inflammation
• mesenchymal stem cells
• migration
• osteoarthritis

Microenvironment plays an important role for stem cell proliferation and differentiation. Macromolecular crowding (MMC) was recently shown to assist stem cells in forming their own matrix microenvironment in vitro. The ability of MMC to support adipose stem cell (ASC) proliferation, metabolism, and multilineage differentiation was studied under different conditions: fetal bovine serum- (FBS-) and human serum- (HS-) based media and xeno- and serum-free (XF/SF) media. Furthermore, the immunophenotype of ASCs under MMC was evaluated. The proliferative capacity of ASCs under MMC was attenuated in each condition. However, osteogenic differentiation was enhanced under MMC, shown by increased deposition of mineralized matrix in FBS and HS cultures. Likewise, significantly greater lipid droplet accumulation and increased collagen IV deposition indicated enhanced adipogenesis under MMC in FBS and HS cultures. In contrast, chondrogenic differentiation was attenuated in ASCs expanded under MMC. The ASC immunophenotype was maintained under MMC with significantly higher expression of CD54. However, MMC impaired metabolic activity and differentiation capacity of ASCs in XF/SF conditions. Both the supportive and inhibitory effects of MMC on ASC are culture condition dependent. In the presence of serum, MMC maintains ASC immunophenotype and enhances adipogenic and osteogenic differentiation at the cost of reduced proliferation.

• Adipose Tissue/cytology
• Cell Differentiation/radiation effects
• Cell Hypoxia
• Cells, Cultured
• Gene Expression Profiling
• Gene Expression Regulation
• Humans
• Hypothermia
• Mesenchymal Stem Cells/cytology
• Mesenchymal Stem Cells/metabolism
• Mesenchymal Stem Cells/radiation effects
• Osteogenesis/genetics
• Osteogenesis/radiation effects
• Vascular Endothelial Growth Factor A/genetics

• DMSO-free cryopreservation
• Human adipose-derived mesenchymal stromal cells
• Platelet lysate
• Sucrose pretreatment
• Xeno-free expansion

Adipose-derived stromal cells (ASCs) are being used extensively in clinical trials. These trials require that ASCs are prepared using good manufacturing practices (GMPs) and are safe for use in humans. The majority of clinical trials in which ASCs are expanded make use of fetal bovine serum (FBS). While FBS is used traditionally in the research setting for in vitro expansion, it does carry the risk of xenoimmunization and zoonotic transmission when used for expanding cells destined for therapeutic purposes. In order to ensure a GMP quality product for cellular therapy, in vitro expansion of ASCs has been undertaken using xeno-free (XF), chemically-defined, and human blood-derived alternatives. These investigations usually include the criteria proposed by the International Society of Cellular Therapy (ISCT) and International Fat Applied Technology Society (IFATS). The majority of studies use these criteria to compare plastic-adherence, morphology, the immunophenotype and the trilineage differentiation of ASCs under the different medium supplemented conditions. Based on these studies, all of the alternatives to FBS seem to be suitable replacements; however, each has its own advantages and drawbacks. Very few studies have investigated the effects of the supplements on the immunomodulation of ASCs; the transcriptome, proteome and secretome; and the ultimate effects in appropriate animal models. The selection of medium supplementation will depend on the downstream application of the ASCs and their efficacy and safety in preclinical studies.

• adipose-derived stromal cells
• fetal bovine serum
• good manufacturing processes
• human serum
• in vitro expansion
• platelet lysate
• platelet poor plasma
• platelet rich plasma

The use of fetal bovine serum (FBS) as a cell culture supplement is discouraged by regulatory authorities to limit the risk of zoonoses and xenogeneic immune reactions in the transplanted host. Additionally, FBS production came under scrutiny due to animal welfare concerns. Platelet derivatives have been proposed as FBS substitutes for the ex-vivo expansion of mesenchymal stem/stromal cells (MSCs) since platelet-derived growth factors can promote MSC ex-vivo expansion. Platelet-derived growth factors are present in platelet lysate (PL) obtained after repeated freezing–thawing cycles of the platelet-rich plasma or by applying physiological stimuli such as thrombin or CaCl₂.

PL-expanded MSCs have been used already in the clinic, taking advantage of their faster proliferation compared with FBS-expanded preparations. Should PL be applied to other biopharmaceutical products, its demand is likely to increase dramatically. The use of fresh platelet units for the production of PL raises concerns due to limited availability of platelet donors. Expired units might represent an alternative, but further data are needed to define safety, including pathogen reduction, and functionality of the obtained PL. In addition, relevant questions concerning the definition of PL release criteria, including concentration ranges of specific growth factors in PL batches for various clinical indications, also need to be addressed. We are still far from a common definition of PL and standardized PL manufacture due to our limited knowledge of the mechanisms that mediate PL-promoting cell growth. Here, we concisely discuss aspects of PL as MSC culture supplement as a preliminary step towards an agreed definition of the required characteristics of PL for the requirements of manufacturers and users.

• Fetal bovine serum
• Manufacturing
• Mesenchymal stem/stromal cells
• Pathogen reduction
• Platelet lysate
• Platelet releasate

• Adipose stem cells
• Bone regeneration
• Calcium phosphates
• Clinical study
• Maxillary sinus floor elevation
• Regenerative medicine

• Adipose Tissue/cytology
• Aged
• Bone Regeneration
• Calcium Phosphates/therapeutic use
• Dental Implants
• Female
• Flow Cytometry
• Humans
• Male
• Maxilla
• Mesenchymal Stem Cell Transplantation
• Mesenchymal Stem Cells/cytology
• Microscopy, Electron, Scanning
• Middle Aged
• Regenerative Medicine/methods
• Sinus Floor Augmentation/methods
• X-Ray Microtomography

• Fibroblast
• Fibrodyplasia ossificans progressiva
• Inflammation
• Ossification
• Osteogenic differentiation
• Transforming growth factor β

• Activin Receptors, Type I/metabolism
• Adolescent
• Adult
• Alkaline Phosphatase/metabolism
• Animals
• Benzamides/pharmacology
• Blood Platelets/metabolism
• Calcification, Physiologic/drug effects
• Calcification, Physiologic/genetics
• Cell Transdifferentiation/drug effects
• Cell Transdifferentiation/genetics
• Cells, Cultured
• Female
• Fibroblasts/drug effects
• Fibroblasts/metabolism
• Fibroblasts/pathology
• Gene Expression Regulation/drug effects
• Humans
• Ki-67 Antigen/metabolism
• Male
• Middle Aged
• Models, Biological
• Myositis Ossificans/genetics
• Myositis Ossificans/metabolism
• Myositis Ossificans/pathology
• Osteogenesis/drug effects
• Osteogenesis/genetics
• Pyrazoles/pharmacology
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• Signal Transduction/drug effects
• Signal Transduction/genetics
• Smad Proteins/metabolism
• Transforming Growth Factor beta/metabolism
• Young Adult

• Adipose-derived stem cells
• Cell proliferation
• Cell therapy
• Culture media
• Phenotype
• Xenogeneic free

• Adipocytes/cytology
• Adipose Tissue/cytology
• Animals
• Blood Platelets/cytology
• Cattle
• Cell Differentiation/drug effects
• Cell Proliferation/drug effects
• Cell- and Tissue-Based Therapy
• Culture Media, Serum-Free/pharmacology
• Flow Cytometry
• Humans
• Regenerative Medicine
• Stem Cells/cytology
• Stem Cells/drug effects

• Biomimetic design
• Biopolymers
• Blood-derived biomaterials
• Extracellular matrix
• Raw materials
• Tissue decellularization
• Transplantation

• R03 DE022838 NIDCR NIH HHS
• R41 DE024343 NIDCR NIH HHS

• Cell therapy
• Endothelial colony-forming progenitor cells
• Human platelet lysate
• Mesenchymal stem cells
• Platelet-rich plasma
• Tissue engineering

Human adipose tissue-derived mesenchymal stem cells (ADMSCs) are considered eligible candidates for cardiovascular stem cell therapy applications due to their cardiac transdifferentiation potential and immunotolerance. Over the years, the in vitro culture of ADMSCs by platelet lysate (PL), a hemoderivate containing numerous growth factors and cytokines derived from platelet pools, has allowed achieving a safe and reproducible methodology to obtain high cell yield prior to clinical administration. Nevertheless, the biological properties of PL are still to be fully elucidated. In this brief report we show the potential ability of PL to induce a permissive state of cardiac-like transdifferentiation and to cause epigenetic modifications. RTPCR results indicate an upregulation of Cx43, SMA, c-kit, and Thy-1 confirmed by immunofluorescence staining, compared to standard cultures with foetal bovine serum. Moreover, PL-cultured ADMSCs exhibit a remarkable increase of both acetylated histones 3 and 4, with a patient-dependent time trend, and methylation at lysine 9 on histone 3 preceding the acetylation. Expression levels of p300 and SIRT-1, two major regulators of histone 3, are also upregulated after treatment with PL. In conclusion, PL could unravel novel biological properties beyond its routine employment in noncardiac applications, providing new insights into the plasticity of human ADMSCs.

In most pre-clinical animal studies investigating stem cell therapy in acute myocardial infarction (AMI), the administered stem cells are isolated from healthy donors. In clinical practice, however, patients who suffer from AMI will receive autologous cells, for example using adipose-derived stem cells (ASC). During AMI, inflammation is induced and we hypothesized that this might affect characteristics of ASC. To investigate this, ASC were isolated from rat adipose tissue 1 day (1D group, n = 5) or 7 days (7D group, n = 6) post-AMI, and were compared with ASC from healthy control rats (Control group, n = 6) and sham-operated rats (Sham 1D group, n = 5). We found that significantly fewer ASC were present 1 day post-AMI in the stromal vascular fraction (SVF), determined by a colony-forming-unit assay (p < 0.001 vs. Control and 7D). These data were confirmed by flow cytometry, showing fewer CD90-positive cells in SVF of the 1D group. When cultured, no differences were found in proliferation rate and cell size between the groups in the first three passages. Also, no difference in the differentiation capacity of ASC was found. In conclusion, it was shown that significantly fewer stem cells were present in the SVF 1 day post-AMI; however, the stem cells that were present showed no functional differences.

• Acute myocardial infarction
• Adipose-derived stem cells
• Mesenchymal stem cells
• Rat acute myocardial infarction model
• Stromal vascular fraction

Administration of mesenchymal stem cells (MSCs) represents a promising treatment option for patients suffering from immunological and degenerative disorders. Accumulating evidence indicates that the healing effects of MSCs are mainly related to unique paracrine properties, opening opportunities for secretome-based therapies. Apart from soluble factors, MSCs release functional small RNAs via extracellular vesicles (EVs) that seem to convey essential features of MSCs. Here we set out to characterize the full small RNAome of MSC-produced exosomes.

We set up a protocol for isolating exosomes released by early passage adipose- (ASC) and bone marrow-MSCs (BMSC) and characterized them via electron microscopy, protein analysis and small RNA-sequencing. We developed a bioinformatics pipeline to define the exosome-enclosed RNA species and performed the first complete small RNA characterization of BMSCs and ASCs and their corresponding exosomes in biological replicates.

Our analysis revealed that primary ASCs and BMSCs have highly similar small RNA expression profiles dominated by miRNAs and snoRNAs (together 64-71 %), of which 150–200 miRNAs are present at physiological levels. In contrast, the miRNA pool in MSC exosomes is only 2-5 % of the total small RNAome and is dominated by a minor subset of miRNAs. Nevertheless, the miRNAs in exosomes do not merely reflect the cellular content and a defined set of miRNAs are overrepresented in exosomes compared to the cell of origin. Moreover, multiple highly expressed miRNAs are precluded from exosomal sorting, consistent with the notion that these miRNAs are involved in functional repression of RNA targets. While ASC and BMSC exosomes are similar in RNA class distribution and composition, we observed striking differences in the sorting of evolutionary conserved tRNA species that seems associated with the differentiation status of MSCs, as defined by Sox2, POU5F1A/B and Nanog expression.

We demonstrate that primary MSCs release small RNAs via exosomes, which are increasingly implicated in intercellular communications. tRNAs species, and in particular tRNA halves, are preferentially released and their specific sorting into exosomes is related to MSC tissue origin and stemness. These findings may help to understand how MSCs impact neighboring or distant cells with possible consequences for their therapeutic usage.

The online version of this article (doi:10.1186/s13287-015-0116-z) contains supplementary material, which is available to authorized users.