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Multipotent mesenchymal stromal/stem cell (MSC) spheroids generated in three-dimensional culture are of considerable interest as a novel therapeutic tool for regenerative medicine. However, the lack of reliable methods for storing MSC spheroids represents a significant roadblock to their successful use in the clinic. An ideal storage medium for MSC spheroids should function as both a vehicle for delivery and a cryoprotectant during storage of spheroids for use at a later time. In this study, we compared the outcomes after subjecting MSC spheroids to a freeze/thaw cycle in three Good Manufacturing Practices-grade cryopreservation media, CryoStor10 (CS10), Stem-Cellbanker (SCB), and Recovery Cell Culture Freezing Media (RFM) or conventional freezing medium (CM) (CM, Dulbecco's modified Eagle's medium containing 20% fetal bovine serum and 10% dimethyl sulfoxide) as a control for 2 months. The endpoints tested were viability, morphology, and expression of stem cell markers and other relevant genes. The results of LIVE/DEAD™ assays and annexin V/propidium iodide staining suggested that viability was relatively higher after one freeze/thaw cycle in CS10 or SCB than after freeze/thaw in CM or RFM. Furthermore, the relative "stemness" and expression of MSC markers were similar with or without freeze/thaw in CS10. Scanning electron microscopy also indicated that the surface morphology of MSC spheroids was well preserved after cryopreservation in CS10. Thus, even though it was tested for a short-term period, we suggest that CS10, which has been approved for clinical use by the U.S. Food and Drug Association, is a promising cryopreservation medium that would facilitate the development of MSC spheroids for future clinical use.

Background Aims: Culture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods, source material and culture media. The purpose of this multicenter study was to assess the impact on MSC expansion, gene expression and other characteristics when different laboratories expanded MSCs from cultures initiated with bone marrow-MSC aliquots derived from the same donor source material yet with different growth media., Methods: Eight centers expanded MSCs using four human platelet lysate (HPL) and one fetal bovine serum (FBS) products as media supplements. The expanded cells were taken through two passages then assessed for cell count, viability, doubling time, immunophenotype, cell function, immunosuppression and gene expression. Results were analyzed by growth media and by center., Results: Center methodologies varied by their local seeding density, feeding regimen, inoculation density, base media and other growth media features (antibiotics, glutamine, serum). Doubling times were more dependent on center than on media supplements. Two centers had appropriate immunophenotyping showing all MSC cultures were positive for CD105, CD73, CD90 and negative for CD34, CD45, CD14, HLA-DR. MSCs cultured in media supplemented with FBS compared with HPL featured greater T-cell inhibition potential. Gene expression analysis showed greater impact of the type of media supplement (HPL versus FBS) than the manufacturing center. Specifically, nine genes were decreased in expression and six increased when combining the four HPL-grown MSCs versus FBS (false discovery rate [FDR] <0.01), however, without significant difference between different sources of HPL (FDR <0.01)., Conclusions: Local manufacturing process plays a critical role in MSC expansion while growth media may influence function and gene expression. All HPL and FBS products supported cell growth., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary, or financial interest in the products or companies described in this article., (Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Introduction: Adipose tissue mesenchymal stem/stromal cells (ASC) can be used as advanced therapy medicinal product in regenerative and cancer medicine. We previously demonstrated Supernatant Rich in Growth Factors (SRGF) can replace fetal bovine serum (FBS) to expand ASC by a clinical grade compliant protocol. The therapeutic potential of ASC is based also on their homing capacity toward inflammatory/cancer sites: oriented cell migration is a fundamental process in this scenario. We investigated the impact of SRGF on ASC migration properties., Methods: The motility/migration potential of ASC expanded in 5% SRGF was analyzed, in comparison to 10% FBS, by standard wound healing, bidimensional chemotaxis and transwell assays, and by millifluidic transwell tests. Mechanisms involved in the migration process were investigated by transient protein overexpression., Results: In comparison to standard 10% FBS, supplementation of the cell culture medium with 5% SRGF, strongly increased migration properties of ASC along the chemotactic gradient and toward cancer cell derived soluble factors, both in static and millifluidic conditions. We showed that, independently from applied migratory stimulus, SRGF expanded ASC were characterized by far lower expression of α-smooth muscle actin (αSMA), a protein involved in the cell migration machinery. Overexpression of αSMA induced a significant and marked decrease in migration capacity of SRGF expanded ASC., Discussion: In conclusion, 5% SRGF addition in the cell culture medium increases the migration potential of ASC, reasonably through appropriate downregulation of αSMA. Thus, SRGF could potentially improve the therapeutic impact of ASC, both as modulators of the immune microenviroment or as targeted drug delivery vehicles in oncology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Agostini, Vicinanza, Lombardi, Da Ros, Marangon, Massarut, Mazzucato and Durante.)

Cold atmospheric pressure plasma (CAP) offers a variety of therapeutic possibilities and induces the formation of reactive chemical species associated with oxidative stress. Mesenchymal stem/stromal cells (MSCs) play a central role in tissue regeneration, partly because of their antioxidant properties and ability to migrate into regenerating areas. During the therapeutic application, MSCs are directly exposed to the reactive species of CAP. Therefore, the investigation of CAP-induced effects on MSCs is essential. In this study, we quantified the amount of ROS due to the CAP activation of the culture medium. In addition, cell number, metabolic activity, stress signals, and migration were analyzed after the treatment of MSCs with a CAP-activated medium. CAP-activated media induced a significant increase in ROS but did not cause cytotoxic effects on MSCs when the treatment was singular and short-term (one day). This single treatment led to increased cell migration, an essential process in wound healing. In parallel, there was an increase in various cell stress proteins, indicating an adaptation to oxidative stress. Repeated treatments with the CAP-activated medium impaired the viability of the MSCs. The results shown here provide information on the influence of treatment frequency and intensity, which could be necessary for the therapeutic application of CAP.

Preparing a suitable cell culture medium that supports the biological needs of the growing cells is crucial to enhancing the success rate of any in vitro and in vivo experiments and minimizing undesirable interferences.  Mesenchymal stem cells ( MSCs) which are powerful regenerative stem cells require being grown in proper culture media to preserve their stemness and therapeutic properties. MSCs are usually grown in Dulbecco's Modified Eagle low glucose Medium (DMEM low glucose) which contains 5.6 mmol/L of glucose and is supplemented with Fetal Bovine Serum (FBS), antibiotics, and 2-Mercaptoethanol. The addition of 2-Mercaptoethanol to the cell culture medium was proposed long ago and has continued to be used until now. Despite the positive effects of adding 2-Mercaptoethanol in the cell culture medium, its use is still controversial and needs continuous updates to limit its interference with experimental treatments. Herein, we found that 2-Mercaptoethanol is beneficial to enhancing the proliferation and survival of MSCs at higher passage numbers while its effect is negligible for earlier passages. This concise study provides updates regarding the suitable time to add 2-Mercaptoethanol which can minimize its intermeddling with the experimental design and treatments.

Bovine serum albumin (BSA) plays a crucial role in cell culture media, influencing cellular processes such as proliferation and differentiation. Although it is commonly included in chondrogenic differentiation media, its specific function remains unclear. This study explores the effect of different BSA concentrations on the chondrogenic differentiation of human adipose-derived stromal/stem cells (hASCs). hASC pellets from six donors were cultured under chondrogenic conditions with three BSA concentrations. Surprisingly, a lower BSA concentration led to enhanced chondrogenesis. The degree of this effect was donor-dependent, classifying them into two groups: (1) high responders, forming at least 35% larger, differentiated pellets with low BSA in comparison to high BSA; (2) low responders, which benefitted only slightly from low BSA doses with a decrease in pellet size and marginal differentiation, indicative of low intrinsic differentiation potential. In all cases, increased chondrogenesis was accompanied by hypertrophy under low BSA concentrations. To the best of our knowledge, this is the first study showing improved chondrogenicity and the tendency for hypertrophy with low BSA concentration compared to standard levels. Once the tendency for hypertrophy is understood, the determination of BSA concentration might be used to tune hASC chondrogenic or osteogenic differentiation.

Among the available therapeutics for the conservative treatment of osteoarthritis (OA), mesenchymal stromal cells (MSCs)-based products appear to be the most promising. Alongside minimally manipulated cell-based orthobiologics, where MSCs are the engine of the bioactive properties, cell expansion under good manufacturing practice (GMP) settings is actively studied to obtain clinical-grade pure populations able to concentrate the biological activity. One of the main characteristics of GMP protocols is the use of clinical-grade reagents, including the recently released serum-free/xeno-free (SFM/XFM) synthetic media, which differ significantly from the traditional reagents like those based on fetal bovine serum (FBS). As SFM/XFM are still poorly characterized, a main lack is the notion of reliable housekeeping genes (HKGs) for molecular studies, either standalone or in combination with standard conditions. Indeed, the aim of this work was to test the stability of five commonly used HKGs ( ACTB , EF1A , GAPDH , RPLP0 , and TBP ) in adipose-derived MSCs (ASCs) cultivated in two commercially available SFM/XFM and to compare outcomes with those obtained in FBS. Four different applets widely recognized by the scientific community (NormFinder, geNorm, comparative ΔCt method, and BestKeeper) were used and data were merged to obtain a final stability order. The analysis showed that cells cultured in both synthetic media had a similar ranking for HKGs stability ( GAPDH being best), albeit divergent from FBS expanded products ( EF1A at top). Moreover, it was possible to identify specific HKGs for side by side studies, with EF1A / TBP being the most reliable normalizers for single SFM/XFM vs. FBS cultured cells and TBP the best one for a comprehensive analysis of all samples. In addition, stability of HKGs was donor-dependent. The normalization effect on selected genes coding for factors known to be involved in OA pathology, and whose amount should be carefully considered for the selection of the most appropriate MSC-based treatment, showed how HKGs choice might affect the perceived amount for the different media or donor. Overall, this work confirms the impact of SFM/XFM conditions on HKGs stability performance, which resulted similarly for both synthetic media analyzed in the study.

Background: Despite a long history of investigation and sustained efforts in clinical testing, the number of market authorisations for mesenchymal stromal cell (MSC) therapies remains limited, with none approved by the United States Food and Drug Administration. Several barriers are impeding the clinical progression of MSC therapies, to the forefront of these is a lack of standardised manufacturing protocols which is further compounded by an absence of biologically meaningful characterisation and release assays. A look at clinical trial registries demonstrates the diversity of MSC expansion protocols with variabilities in cell source, isolation method and expansion medium, among other culture variables, making it extraordinarily difficult to compare study outcomes. Current identification and characterisation standards are insufficient; they are not specific to MSCs and do not indicate cell function or therapeutic action., Methods: This work analysed the influence of five widely used culture media formulations on the colony-forming potential, proliferation kinetics, trilineage differentiation potential and immunomodulatory potential of human bone marrow-derived MSCs (BM-MSCs). The surface marker expression profiles were also characterised using a high-content flow cytometry screening panel of 243 markers., Results: Significant differences in the biological attributes of BM-MSCs including clonogenicity, proliferation, differentiation propensity and immunomodulatory capacity were revealed in response to the composition of the culture medium. Despite their biological differences, all cell preparations uniformly and strongly expressed the standard positive markers proposed for BM-MSCs: CD73, CD90 and CD105. Immunophenotypic profiling revealed that the culture medium also had a significant influence on the surface proteome, with one-third of tested markers exhibiting variable expression profiles. Principal component analysis demonstrated that BM-MSCs isolated and expanded in a proprietary xeno- and serum-free medium displayed the most consistent cell phenotypes with little variability between donors compared to platelet lysate and foetal bovine serum-containing media., Conclusions: These data suggest that media composition has a highly significant impact on the biological attributes of MSCs, but standard surface marker tests conceal these differences. The results indicate a need for (1) standardised approaches to manufacturing, with an essential focus on defined media and (2) new biologically relevant tests for MSC characterisation and product release., (© 2023. The Author(s).)

We focus on this study in designing an alternative technique for obtaining mesenchymal stem cells (MSCs) from residual tissue, Hoffa fat, in arthroscopic procedures. Two males and two females were included, and underwent knee arthroscopy; a sample of infrapatellar adipose tissue was obtained with basket forceps. The primary culture was made using the explant method and the culture media: DMEM-high glucose, supplemented with 10% of inactivated human allogeneic serum. All the cellular cultures remained under culture conditions for three weeks, after that by flow cytometry the cells were characterized by MSCs antibody panel: CD105, CD73 and CD90. Subsequently, in the first pass, the MSCs were cultured in commercial human chondrogenic, osteogenic and adipogenic mediums, respectively. After primary culture, we obtained on average 95,600.00 ± 7,233.26 cells/cm2, and the duplication time of MSCs isolate from Hoffa fat pad was established in 39 hours. By flow cytometry, we found that surface markers percentage for expanded MSCs (CD105, CD73, CD90) in primary culture significantly increased and its morphology was fibroblastic-like. After differentiation culture which was made in the first pass, by immunofluorescence, we obtained positive cell markers for three lineages of differentiation, adipocytes: LPL protein, osteocytes: RUNX2, Osteopontin, chondrocytes: SOX9, Aggrecan and COL2A1. We managed to isolate a significant number of MSCs from this source using an easy method to implement and minimal nutrient supplementation, with high potential for differentiation to mature mesenchymal tissues and potential use in basic experimental, preclinical and even clinical research.

Therapies utilizing autologous mesenchymal cell delivery are being investigated as anti-inflammatory and regenerative treatments for a broad spectrum of age-related diseases, as well as various chronic and acute pathological conditions. Easily available allogeneic full-term human placenta mesenchymal stromal cells (pMSCs) were used as a potential pro-regenerative, cell-based therapy in degenerative diseases, which could be applied also to elderly individuals. To explore the potential of allogeneic pMSCs transplantation for pro-regenerative applications, such cells were isolated from five different term-placentas, obtained from the dissected maternal, endometrial (mpMSCs), and fetal chorion tissues (fpMSCs), respectively. The proliferation rate of the cells in the culture, as well as their shape, in vitro differentiation potential, and the expression of mesenchymal lineage and stem cell markers, were investigated. Moreover, we studied the expression of immune checkpoint antigen CD276 as a possible modulation of the rejection of transplanted non-HLA-matched homologous or even xeno-transplanted pMSCs. The expression of the cell surface markers was also explored in parallel in the cryosections of the relevant intact placenta tissue samples. The expansion of pMSCs in a clinical-grade medium complemented with 5% human platelet lysate and 5% human serum induced a significant expression of CD276 when compared to mpMSCs expanded in a commercial medium. We suggest that the expansion of mpMSCs, especially in a medium containing platelet lysate, elevated the expression of the immune-regulatory cell surface marker CD276. This may contribute to the immune tolerance towards allogeneic pMSC transplantations in clinical situations and even in xenogenic animal models of human diseases. The endurance of the injected comparably young human-term pMSCs may promote prolonged effects in clinical applications employing non-HLA-matched allogeneic cell therapy for various degenerative disorders, especially in aged adults.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) has been shown to rely on their immunomodulatory and regenerative properties. In order to obtain sufficient numbers of cells for clinical applications, MSCs have to be expanded ex vivo. Expansion media with xenogeneic-free (XF) growth-promoting supplements like human platelet lysate (PL) or serum- and xenogeneic-free (SF/XF) formulations have been established as safe and efficient, and both groups provide different beneficial qualities. In this study, MSCs were expanded in XF or SF/XF media as well as in mixtures thereof. MSCs cultured in these media were analyzed for phenotypic and functional properties. MSC expansion was optimal with SF/XF conditions when PL was present. Metabolic patterns, consumption of growth factors, and secretome of MSCs differed depending on the type and concentration of supplement. The lactate per glucose yield increased along with a higher proportion of PL. Many factors in the supernatant of cultured MSCs showed distinct patterns depending on the supplement (e.g., FGF-2, TGFβ, and insulin only in PL-expanded MSC, and leptin, sCD40L PDGF-AA only in SF/XF-expanded MSC). This also resulted in changes in cell characteristics like migratory potential. These findings support current approaches where growth media may be utilized for priming MSCs for specific therapeutic applications.

Development of clinical-grade, cell preparations is central to cGMP (good manufacturing practice compliant) conditions. This study aimed to investigate the potential of two serum/xeno-free, cGMP (StemPro, StemMacs) culture media to maintain "stemness" of human minor salivary gland stem cell (mSG-SC) cultures compared to a complete culture medium (CCM). Overall, StemMacs resulted in higher proliferation rates after p.6 compared to the conventional serum-based medium, while StemPro showed substantial delays in cell proliferation after p.9. The mSG-SCs cultures exhibited two distinct cell populations at early passages a mesenchymal subpopulation and an epithelial-like subpopulation. Expression of several markers (CD146, STRO-1, SSEA-4, CD105, CD106, CD34, K 7/8, K14, K18) variably decreased with prolonged passaging (all three media). The percentage of SA-β-gal positive cells was initially higher for StemMacs compared to StemPro/CCM and increased with prolonged passaging in all cases. The telomere fragment length decreased with prolonged passaging in all three media but more pronouncedly for the CCM. Expansion under serum-free conditions caused pronounced upregulation of ALP and BMP-2, with parallel complete elimination of the baseline expressions of LPL (all three media) and ACAN (serum-free media), therefore, showing a preferential shift of the mSG-SCs towards osteogenic phenotypes. Finally, several markers (Nanog, SOX-2, PDX-1, OTX2, GSC, HCG) decreased with prolonged culture, indicating successive loss of "stemness". Based on the findings, it seems that StemPro preserve stemness of the mSG-SCs after prolonged culture. Nevertheless, there is still a vacant role for the ideal development of clinical-grade culture conditions., (© 2023. The Author(s).)

The intrinsic healing following tendon injury is ideal, in which tendon progenitor cells proliferate and migrate to the injury site to directly bridge or regenerate tendon tissue. However, the mechanism determining why and how those cells are attracted to the injury site for tendon healing is not understood. Since the tenocytes near the injury site go through apoptosis or necrosis following injury, we hypothesized that secretions from injured tenocytes might have biological effects on cell proliferation and migration to enhance tendon healing. Tenocyte apoptosis was induced by 24 h cell starvation. Apoptotic body-rich media (T-ABRM) and apoptotic body-depleted media (T-ABDM) were collected from culture media after centrifuging. Tenocytes and bone marrow-derived stem cells (BMDSCs) were isolated and cultured with the following four media: (1) T-ABRM, (2) T-ABDM, (3) GDF-5, or (4) basal medium with 2% fetal calf serum (FCS). The cell activities and functions were evaluated. Both T-ABRM and T-ABDM treatments significantly stimulated the cell proliferation, migration, and extracellular matrix synthesis for both tenocytes and BMDSCs compared to the control groups (GDF-5 and basal medium). However, cell proliferation, migration, and extracellular matrix production of T-ABRM-treated cells were significantly higher than the T-ABDM, which indicates the apoptotic bodies are critical for cell activities. Our study revealed the possible mechanism of the intrinsic healing of the tendon in which apoptotic bodies, in the process of apoptosis, following tendon injury promote tenocyte and stromal cell proliferation, migration, and production. Future studies should analyze the components of the apoptotic bodies that play this role, and, thus, the targeting of therapeutics can be developed.

Background: Mesenchymal stem cells (MSCs) have been suggested as an appropriate source for diabetes cell-based therapies. The high proliferation and differentiation capacity of fetal MSCs and the role of fetal pancreatic-derived MSCs (FPMSCs) in islet generation make them good candidates for diabetes treatment. To manufacture clinical-grade MSCs, animal-free culture protocols are preferred. The current study aimed to establish a xeno-free/GMP-compliant protocol for FPMSCs manufacturing. The focus was on the effects of fetal bovine serum (FBS) replacement with pooled human serum (HS)., Material and Methods: FPMSCs were isolated and expanded from the pancreas of legally aborted fetuses with few modifications in our previously established protocol. The cells were expanded in two different culture media, including DMEM supplemented with 10% FBS or 10% pooled HS. A side-by-side comparison was made to evaluate the effect of each serum on proliferation rate, cell cycle, senescence, multi-lineage differentiation capacity, immunophenotype, and tumorigenesis of FPMSCs., Results: Flow cytometry analysis and three-lineage differentiation ability demonstrated that fibroblast-like cells obtained from primary culture had MSCs' characteristics. The FPMSCs displayed similar morphology and CD markers expression in both sera. HS had a higher proliferative effect on FPMSCs than FBS. In FBS, the cells reached senescence earlier. In addition to normal karyotypes and anchorage-dependent growth, in vivo tumor formation was not seen., Conclusion: Our results demonstrated that HS was a better serum alternative than FBS for in vitro expansion of FPMSCs. Compared with FBS, HS increased FPMSCs' proliferation rate and decreased their senescence. In conclusion, HS can effectively replace FBS for clinical-grade FPMSCs manufacturing., (© 2022. The Author(s).)

The osteogenic differentiation of mesenchymal stem cells is now a standard procedure in modern bone tissue engineering. As this is a promising field for future clinical applications, many cell culture media exist to promote osteogenic differentiation. Prior to differentiation, cells must be expanded to obtain sufficient numbers for experiments. Little evidence is available regarding the optimal media combination for expansion and differentiation to maximize the osteogenic response. Therefore, human BM-MSCs ( n = 6) were expanded in parallel in DMEM (Dulbecco's Modified Eagle Medium) LG (Low Glucose) and α-MEM (Minimum Essential Media alpha-modification), followed by simultaneous monolayer differentiation toward the osteogenic lineage in: 1. DMEM LG (Low Glucose), 2. DMEM HG (High Glucose), 3. α-MEM, 4. "Bernese medium", and 5. "Verfaillie medium", with a corresponding negative control (total 20 groups). As a marker for osteogenic differentiation, hydroxyapatite was accessed using radioactive 99m Tc-HDP labeling and quantitative alizarin red staining. The results indicate that all media except "Bernese medium" are suitable for osteogenic differentiation, while there was evidence that DMEM LG is partly superior when used for expansion and differentiation of BM-hMSCs. Using "Verfaillie medium" after DMEM LG expansion led to the highest grade of osteogenic differentiation. Nevertheless, the difference was not significant. Therefore, we recommend using DMEM LG for robust osteogenic differentiation, as it is highly suitable for that purpose, economical compared to other media, and requires little preparation time.

Mesenchymal stem cell (MSC) culturing for cell therapies needs a step forward to be routinely used in clinical settings. Main concerns regard the use of animal origin reagents, in particular supplementing the culture medium with FBS. Lately, Human Platelet Lysate (HPL) has been proposed as animal-free alternative, described as an excellent supplement for culturing MSCs. The aim of this systematic review was to analyze the current literature on the effect of HPL and FBS on ASCs and BMSCs. The primary outcome was the proliferation rate of cells cultured with FBS and HPL. Differences in terms of doubling time (DT) and population doubling (PD) were evaluated by meta-analysis, subgrouping data according to the cell type. A total of 35 articles were included. BMSCs and ASCs were used in 65.7% (23) and 28.6% (10) studies, respectively. Only two studies included both cell types. Overall, 22 studies were eligible for the meta-analysis. Among them, 9 articles described ASCs and 13 BMSCs. The results showed that BMSCs and ASCs cultured with 10% HPL and 5% HPL have lower DT and higher PD compared to cells cultured with 10% FBS. A possible correlation between the DT decrease and the application of at least 3 freeze/thaw cycles to induce platelet lysis was found. Additionally, HPL increased VEGF secretion and maintained the immuno-modulatory abilities for both cell types. The clarification reported here of the higher efficiency of HPL compared to FBS can help the transition of the scientific community towards clinical-related procedures. 1. The meta-analysis shows that HPL induces a population doubling increase and a doubling time decrease of both ASCs and BMSCs compared to FBS. 2. When at least 3 freeze/thaw cycles are applied to induce platelet lysis, the doubling time of HPL-cultured cells is lower than FBS-cultured cells (Created with BioRender.com)., (© 2022. The Author(s).)

Objective: The study is aimed to verify the possibility of using humanized alternatives to fetal bovine serum (FBS) such as umbilical cord blood plasma (CBP) and AB + plasma to support the long-term growth of mesenchymal stromal cells (MSCs) derived from the umbilical cord. We hypothesized that umbilical CBP would be a potential substitute to FBS, especially for small scale autologous clinical transplantations., Methods: The MSCs were cultured for six consecutive passages to evaluate xeno-free media's ability to support long-term growth. Cell proliferation rates, colony-forming-unit (CFU) efficiency and population doublings of expanded MSCs, were investigated. Ex vivo expanded MSCs were further characterized using flow cytometry and quantitative PCR. The impact of cryopreservation and composition of cryomedium on phenotype, viability of MSC was also assessed., Results: Our results on cell proliferation, colony-forming unit efficiency suggested that the expansion of the cells was successfully carried out in media supplemented with humanized alternatives. MSCs showed lower CFU counts in FBS (~ 25) than humanized alternatives (~ 35). The gene expression analysis revealed that transcripts showed significant differential expression by two to three folds in the FBS group compared with MSCs grown in medium with humanized alternatives (p < 0.05). In addition, MSCs grown in a medium with FBS had more osteogenic activity, a signature of unwanted differentiation. The majority of ex vivo expanded MSCs at early and late passages expressed CD44 + , CD73 + , CD105 + , CD90 + , and CD166 + in all the experimental groups tested (~ 90%). In contrast to the other MSC surface markers, expression levels of STRO-1 + (~ 21-10%) and TNAP + (~ 29-11%) decreased with the increase in passage number for MSCs cultured in a FBS-supplemented medium (p < 0.05)., Conclusion: Our results established that CBP supported culture of umbilical cord tissue-derived MSCs and is a safer Xeno free replacement to FBS. The use of CBP also enables the storage of umbilical cord tissue derived MSCs in patient-specific conditions to minimize adverse events if cells are delivered directly to the patient., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

It is well known that extracellular matrix stiffness can affect cell fate and change dynamically during many biological processes. Existing experimental means for in situ matrix stiffness modulation often alters its structure, which could induce additional undesirable effects on cells. Inspired by the phenomenon of depth sensing by cells, we introduce here core-shell microfibers with a thin collagen core for cell growth and an alginate shell that can be dynamically stiffened to deliver mechanical stimuli. This allows for the maintenance of biochemical properties and structure of the surrounding microenvironment, while dynamically modulating the effective modulus "felt" by cells. We show that simple addition of Sr 2+ in media can easily increase the stiffness of initially Ca 2+ cross-linked alginate shells. Thus, despite the low stiffness of collagen cores (<5 kPa), the effective modulus of the matrix "felt" by cells are substantially higher, which promotes osteogenesis differentiation of human mesenchymal stem cells. We show this effect is more prominent in the stiffening microfiber compared to a static microfiber control. This approach provides a versatile platform to independently and dynamically modulate cellular microenvironments with desirable biochemical, physical, and mechanical stimuli without an unintended interplay of effects, facilitating investigations of a wide range of dynamic cellular processes.

In the field of regenerative medicine, considerable advances have been made from the technological and biological point of view. However, there are still large gaps to be filled regarding translation and application of mesenchymal stromal cell (MSC)-based therapies into clinical practice. Indeed, variables such as cell type, unpredictable donor variation, and expansion/differentiation methods lead to inconsistencies. Most protocols use bovine serum (FBS) derivatives during MSC expansion. However, the xenogeneic risks associated with FBS limits the use of MSC-based products in clinical practice. Herein we compare a chemically defined, xenogeneic-free commercial growth medium with a conventional medium containing 10% FBS and 5 ng/ml FGF2. Furthermore, the effect of a fibronectin-coated growth surface was investigated. The effect of the different culture conditions on chondrogenic commitment was assessed by analyzing matrix deposition and gene expression of common chondrogenic markers. Chondrogenic differentiation potential was similar between the FBS-containing αMEM and the chemically defined medium with fibronectin coating. On the contrary, the use of fibronectin coating with FBS-containing medium appeared to reduce the differentiation potential of MSCs. Moreover, cells that were poorly responsive to in vitro chondrogenic stimuli were shown to improve their differentiation potential after expansion in a TGF-β1 containing medium. In conclusion, the use of a xenogeneic-free medium provides a suitable alternative for human bone marrow MSC expansion, due the capability to maintain cell characteristic and potency. To further improve chondrogenic potential of BMSCs, priming the cells with TGF-β1 during expansion is a promising strategy.

Human oral mucosa stem cells (hOMSCs) arise from the neural crest, they can self-renew, proliferate, and differentiate to several cell lines and could represent a good source for application in tissue engineering. Because of their anatomical location, hOMSCs are easy to isolate, have multilineage differentiation capacity and express embryonic stem cells markers such as-Sox2, Oct3/4 and Nanog. We have used SHEM (supplemented hormonal epithelial medium) media and cultured hOMSCs over human amniotic membrane and determined the cell's capacity to differentiate to an epithelial-like phenotype and to express corneal specific epithelial markers-CK3, CK12, CK19, Pan-cadherin and E-cadherin. Our results showed that hOMSCs possess the capacity to attach to the amniotic membrane and express CK3, CK19, Pan-Cadherin and E-Cadherin without induction with SHEM media and expressed CK12 or changed the expression pattern of E-Cadherin to a punctual-like feature when treated with SHEM media. The results observed in this study show that hOMSCs possess the potential to differentiate toward epithelial cells. In conclusion, our results revealed that hOMSCs readily express markers for corneal determination and could provide the ophthalmology field with a therapeutic alternative for tissue engineering to achieve corneal replacement when compared with other techniques. Nevertheless, further studies are needed to develop a predictable therapeutic alternative for cornea replacement.

Previous studies have shown that mesenchymal stem cells (MSCs) derived from various tissue sources can be differentiated into smooth muscle-like cells (SMLCs) in vitro. In this paper, dental pulp-derived mesenchymal stem cells (DPSCs) were evaluated for their differentiation ability towards smooth muscle-like cells (SMLCs) under the effect of widely used cytokines (TGF- β 1 and PDGF-BB) with special focus on different culturing environments. For this purpose, both the commercially used culturing plates (Norm-c) and 0.1% gelatin-precoated (Gel-c) plates were used. Isolated cells displayed plastic adherence, pluripotency and cell surface marker profiling, and adipogenic and osteogenic differentiation potential with lineage specific marker expression. Differentiated cells induced under different culturing plates showed successful differentiation into SMLCs by positively expressing smooth muscle cell (SMC) specific markers both at the mRNA and protein levels. Gelatin coating could substantially enhance DPSC differentiation potential than Norm-c-induced cells. However, the absence of mature marker MHY-11 by immunostaining results from all treatment groups further indicated the development of immature and synthetic SMLCs. Finally, it was concluded that DPSC differentiation ability into SMLCs can be enhanced under cytokine treatment as well as by altering the cellular niche by precoating the culturing plates with suitable substrates. However, to get fully functional, contractile, and mature SMLCs, still many different cytokine cocktail combinations and more suitable coating substrates will be needed., Competing Interests: The authors do not have conflict of interests regarding the current work., (Copyright © 2021 Jinhee Ha et al.)

The aim of this study was to differentiate canine adipose-derived mesenchymal stem cells (ADMSCs) into insulin-producing cells by using culture media with different compositions to determine the most efficient media. Stem cells isolated from the fat tissues close to the bitch uterus were distributed into 6 groups: (1) Dulbecco's modified Eagle medium (DMEM)-high glucose (HG), β-mercaptoethanol, and nicotinamide; (2) DMEM-HG, β-mercaptoethanol, nicotinamide, and exendin-4; (3) DMEM-HG, β-mercaptoethanol, nicotinamide, exendin-4, B27, nonessential amino acids, and l-glutamine; (4) DMEM-HG, β-mercaptoethanol, and nicotinamide (for the initial 8-d period), and DMEM-HG, β-mercaptoethanol, nicotinamide, exendin-4, B27, nonessential amino acids, l-glutamine, and basic fibroblast growth factor (for the remaining 8-d period); (5) DMEM-HG and fetal bovine serum; and (6) DMEM-low glucose and fetal bovine serum (standard control group). Adipose-derived mesenchymal stem cells from groups 1 to 5 gradually became round in shape and gathered in clusters. These changes differed between the groups. In group 3, the cell clusters were apparently more in numbers and gathered as bigger aggregates. Dithizone staining showed that groups 3 and 4 were similar in terms of the mean area of each aggregate stained for insulin. However, only in group 4, the number of insulin aggregates and the total area of aggregates stained were significantly bigger than in the other groups. The mRNA expression of PDX1, BETA2, MafA, and Insulin were also confirmed in all the groups. We conclude that by manipulating the composition of the culture medium it is possible to induce canine ADMSCs into insulin-producing cells, and the 2-staged protocol that was used promoted the best differentiation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The growth and proliferation of mesenchymal stem cells are very sensitive in in vitro and a number of factors like media play a significant role in that context. In this study we assessed effect of different media on growth and proliferation of bone marrow derived mesenchymal stem cells (BMMSCs). The BMMSCs were isolated from caprine bone marrow and were subjected to magnetic activated cell sorting against CD90 + , CD105 + , CD271 + and CD34 - along with FC blocker. After characterisation, 2 × 10 4 cells were seeded in 12 well culture plates in four different media viz. MesenCult, MesenPRO, StemPro and complete DMEM (15 % FBS) to study their growth kinetic for 6 days from passage 0 (P0) to passage 3 (P3). The population doubling time (PDT) was derived from growth curve using logarithmic formula. The results showed that the BMMSCs growth and proliferation was highest in MesenCult media in P0 which varied significantly (p < 0.05) from rest of media and from P1 to P3, it was MesenPRO which yielded maximum cells (p < 0.05). The PDT was also in line with growth curve findings. In conclusion, the MesenPRO media had higher growth and proliferation rate from P1 to P3 although MesenCult had higher cell numbers in P0. In conclusion, the use of MesenPRO media could be a better option than conventional media when mesenchymal stem cells are used in clinical applications and other therapeutic purposes taking consideration to its higher growth and proliferation rate. And MesenCult would be a great option to harvest MSCs from P0., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background Aims: Mesenchymal stem/stromal cell (MSC)-based therapies have gained attention as potential alternatives for multiple musculoskeletal indications based on their trophic and immunomodulatory properties. The infrapatellar fat pad (IFP) serves as a reservoir of MSCs, which play crucial roles modulating inflammatory and fibrotic events at the IFP and its neighboring tissue, the synovium. In an effort to comply with the existing regulatory framework regarding cell-based product manufacturing, we interrogated the in vitro immunomodulatory capacity of human-derived IFP-MSCs processed under different conditions, including a regulatory-compliant protocol, in addition to their response to the inflammatory and fibrotic environments often present in joint disease., Methods: Immunophenotype, telomere length, transcriptional and secretory immunomodulatory profiles and functional immunopotency assay were assessed in IFP-MSCs expanded in regular fetal bovine serum (FBS)-supplemented medium and side-by-side compared with same-donor cells processed with two media alternatives (i.e., regulatory-compliant pooled human platelet lysate [hPL] and a chemically reinforced/serum-reduced [Ch-R] formulation). Finally, to assess the effects of such formulations on the ability of the cells to respond to pro-inflammatory and pro-fibrotic conditions, all three groups were stimulated ex vivo (i.e., cell priming) with a cocktail containing TNFα, IFNγ and connective tissue growth factor (tumor-initiating cells) and compared with non-induced cohorts assessing the same outcomes., Results: Non-induced and primed IFP-MSCs expanded in either hPL or Ch-R showed distinct morphology in vitro, similar telomere dynamics and distinct phenotypical and molecular profiles when compared with cohorts grown in FBS. Gene expression of IL-8, CD10 and granulocyte colony-stimulating factor was highly enriched in similarly processed IFP-MSCs. Cell surface markers related to the immunomodulatory capacity, including CD146 and CD10, were highly expressed, and secretion of immunomodulatory and pro-angiogenic factors was significantly enhanced with both hPL and Ch-R formulations. Upon priming, the immunomodulatory phenotype was enhanced, resulting in further increase in CD146 and CD10, significant CXCR4 presence and reduction in TLR3. Similarly, transcriptional and secretory profiles were enriched and more pronounced in IFP-MSCs expanded in either hPL or Ch-R, suggesting a synergistic effect between these formulations and inflammatory/fibrotic priming conditions. Collectively, increased indoleamine-2,3-dioxygenase activity and prostaglandin E2 secretion for hPL- and Ch-R-expanded IFP-MSCs were functionally reflected by their robust T-cell proliferation suppression capacity in vitro compared with IFP-MSCs expanded in FBS, even after priming., Conclusions: Compared with processing using an FBS-supplemented medium, processing IFP-MSCs with either hPL or Ch-R similarly enhances their immunomodulatory properties, which are further increased after exposure to an inflammatory/fibrotic priming environment. This evidence supports the adoption of regulatory-compliant practices during the manufacturing of a cell-based product based on IFP-MSCs and anticipates a further enhanced response once the cells face the pathological environment after intra-articular administration. Mechanistically, the resulting functionally enhanced cell-based product has potential utilization as a novel, minimally invasive cell therapy for joint disease through modulation of local immune and inflammatory events., (Copyright © 2020 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background Aims: Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues., Methods: MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium., Results: Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected., Conclusions: The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro., (Copyright © 2020 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Using multiplex analysis, we performed a comparative study of cytokine and growth factor production by human umbilical cord tissue-derived multipotent mesenchymal stromal cells (UC-MSC) cultured under standard conditions and in the presence of human umbilical cord blood serum (UCBS). It was found that the secretion of most studied molecules, including well-known inductors of regeneration HGF, G-CSF, GM-CSF, and VEGF by UCMSC considerably increased in the presence of 5% UCBS. The use of UCBS allows not only obtaining xenogenic-free cellular and cell-free therapeutic products, but also increasing the secretion of most biologically active molecules capable of stimulating repair processes.

Background Aims: Human platelet lysate can replace fetal bovine serum (FBS) for xeno-free ex vivo expansion of mesenchymal stromal cells (MSCs), but pooling of platelet concentrates (PCs) increases risks of pathogen transmission. We evaluated the feasibility of performing nanofiltration of platelet lysates and determined the impact on expansion of bone marrow-derived MSCs., Methods: Platelet lysates were prepared by freeze-thawing of pathogen-reduced (Intercept) PCs suspended in 65% storage solution (SPP+) and 35% plasma, and by serum-conversion of PCs suspended in 100% plasma. Lysates were added to the MSC growth media at 10% (v/v), filtered and subjected to cascade nanofiltration on 35- and 19-nm Planova filters. Media supplemented with 10% starting platelet lysates or FBS were used as the controls. Impacts of nanofiltration on the growth media composition, removal of platelet extracellular vesicles (PEVs) and MSC expansion were evaluated., Results: Nanofiltration did not detrimentally affect contents of total protein and growth factors or the biochemical composition. The clearance factor of PEVs was >3 log values. Expansion, proliferation, membrane markers, differentiation potential and immunosuppressive properties of cells in nanofiltered media were consistently better than those expanded in FBS-supplemented media. Compared with FBS, chondrogenesis and osteogenesis genes were expressed more in nanofiltered media, and there were fewer senescent cells over six passages., Conclusions: Nanofiltration of growth media supplemented with two types of platelet lysates, including one prepared from pathogen-reduced PCs, is technically feasible. These data support the possibility of developing pathogen-reduced xeno-free growth media for clinical-grade propagation of human cells., (Copyright © 2020 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Objective: Rats are an early preclinical model for cartilage tissue engineering, and a practical species for investigating the effects of aging. However, rats may be a poor aging model for mesenchymal stem cells (MSCs) based on laboratory reports of a severe decline in chondrogenesis beyond young adulthood. Such testing has not been conducted with MSCs seeded in a scaffold, which can improve the propensity of MSCs to undergo chondrogenesis. Therefore, the objective of this study was to evaluate chondrogenesis of middle-aged rat MSCs encapsulated in agarose., Design: MSCs from 14- to 15-month-old rats were expanded, seeded into agarose, and cultured in chondrogenic medium with or without 5% serum for 15 days. Samples were evaluated for cell viability and cartilaginous extracellular matrix (ECM) accumulation. Experiments were repeated using MSCs from 6-week-old rats., Results: During expansion, middle-aged rat MSCs demonstrated a diminishing proliferation rate that was improved ~2-fold in part by transient exposure to chondrogenic medium. In agarose culture in defined medium, middle-aged rat MSCs accumulated ECM to a much greater extent than negative controls. Serum supplementation improved cell survival ~2-fold, and increased ECM accumulation ~3-fold. Histological analysis indicated that defined medium supported chondrogenesis in a subset of cells, while serum-supplementation increased the frequency of chondrogenic cells. In contrast, young rat MSCs experienced robust chondrogenesis in defined medium that was not improved with serum-supplementation., Conclusions: These data demonstrate a previously-unreported propensity of middle-aged rat MSCs to undergo chondrogenesis, and the potential of serum to enhance chondrogenesis of aging MSCs.

A previous study identified kartogenin (KGN) as a potent modulator of bone marrow mesenchymal stem/stromal cell (BMSC) chondrogenesis. This initial report did not contrast KGN directly against transforming growth factor-beta 1 (TGF-β1), the most common growth factor used in chondrogenic induction medium. Herein, we directly compared the in vitro chondrogenic potency of TGF-β1 and KGN using a high resolution micropellet model system. Micropellets were cultured for 7-14 days in medium supplemented with TGF-β1, KGN, or both TGF-β1 + KGN. Following 14 days of induction, micropellets exposed to TGF-β1 alone or TGF-β1 + KGN in combination were larger and produced more glycosominoglycan (GAG) than KGN-only cultures. When TGF-β1 + KGN was used, GAG quantities were similar or slightly greater than the TGF-β1-only cultures, depending on the BMSC donor. BMSC micropellet cultures supplemented with KGN alone contracted in size over the culture period and produced minimal GAG. Indicators of hypertrophy were not mitigated in TGF-β1 + KGN cultures, suggesting that KGN does not obstruct BMSC hypertrophy. KGN appears to have weak chondrogenic potency in human BMSC cultures relative to TGF-β1, does not obstruct hypertrophy, and may not be a viable alternative to growth factors in cartilage tissue engineering.

Cell sheet technology exploits temperature responsive cell culture dishes (TRCDs) as versatile cell harvesting methods to yield contiguous cell monolayers robustly held together by cell-cell junctions, receptors, and endogenous extracellular matrix. More than 15 years of clinical data using autologous-sourced cell sheets demonstrate enhanced therapeutic properties through increased cell retention at target tissue sites. Recently, several preclinical studies have also been reported using mesenchymal stem cell (MSC) sheets in wound healing, cardiac ischemia therapies, and pancreatic regeneration. However, optimized MSC sheet fabrication conditions have not yet been reported. In this study, we identified specific conditions for reliable human MSC sheet fabrication by comparing cell growth media supplements (fetal bovine serum [FBS] and human platelet lysate [hPL]). Human umbilical cord-derived MSCs cultured in FBS and hPL exhibit different actin cytoskeletal structures related to their cell morphologies and adhesion. MSCs cultured in FBS media showed stable cell adhesion on TRCDs with flattened cell shapes and aligned actin cytoskeletal structure. This stable cell adhesion enables production of consistent MSC cell sheets, with controlled cell sheet detachment. Conversely, cell sheet fabrication in hPL media exhibits poor reproducibility being more sensitive to temperature- and culture time-induced release due to weak cell adhesion. These findings suggest that stable MSC adhesion to TRCDs is important to reliable MSC sheet fabrication methods and that MSC growth media supplementation directly affects cell adhesion during culture., (© 2020 John Wiley & Sons, Ltd.)

Background: Clinical application of mesenchymal stromal cells (MSCs) usually requires an in vitro expansion step to reach clinically relevant numbers. In vitro cell expansion necessitates supplementation of basal mammalian cell culture medium with growth factors. To avoid using supplements containing animal substances, human platelet lysates (hPL) produced from expired and pathogen inactivated platelet concentrates can be used in place of fetal bovine serum. However, globally, most transfusion units are currently not pathogen inactivated. As blood banks are the sole source of platelet concentrates for hPL production, it is important to ensure product safety and standardized production methods. In this proof-of-concept study we assessed the feasibility of producing hPL from expired platelet concentrates with pathogen inactivation applied after platelet lysis by evaluating the retention of growth factors, cytokines, and the ability to support MSC proliferation and tri-lineage differentiation., Methodology/principal Findings: Bone marrow-derived MSCs (BM-MSCs) were expanded and differentiated using hPL derived from pathogen inactivated platelet lysates (hPL-PIPL), with pathogen inactivation by amotosalen/ultraviolet A treatment applied after lysis of expired platelets. Results were compared to those using hPL produced from conventional expired pathogen inactivated platelet concentrates (hPL-PIPC), with pathogen inactivation applied after blood donation. hPL-PIPL treatment had lower concentrations of soluble growth factors and cytokines than hPL-PIPC treatment. When used as supplementation in cell culture, BM-MSCs proliferated at a reduced rate, but more consistently, in hPL-PIPL than in hPL-PIPC. The ability to support tri-lineage differentiation was comparable between lysates., Conclusion/significance: These results suggest that functional hPL can be produced from expired and untreated platelet lysates by applying pathogen inactivation after platelet lysis. When carried out post-expiration, pathogen inactivation may provide a valuable solution for further standardizing global hPL production methods, increasing the pool of starting material, and meeting future demand for animal-free supplements in human cell culturing., Competing Interests: OES and SJM are are shareholders in Platome biotechnology (PB). SJM is the CEO of PB and recived salaries from PB during the study. CC holds no shares and was not paid by BP during the study. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Introduction: The olfactory neuro-epithelium has an intrinsic capability of renewal during lifetime provided by the existence of globose and horizontal olfactory precursor cells. Additionally, mesenchymal stromal olfactory cells also support the homeostasis of the olfactory mucosa cell population. Under in vitro culture conditions with Dulbecco modified eagle/F12 medium supplemented with 10% fetal bovine serum, tissue biopsies from upper turbinate have generated an adherent population of cells expressing mainly mesenchymal stromal phenotypic markers. A closer examination of these cells has also found co-expression of olfactory precursors and ensheathing cell phenotypic markers. These results were suggestive of a unique property of olfactory mesenchymal stromal cells as potentially olfactory progenitor cells., Objective: To study whether the expression of these proteins in mesenchymal stromal cells is modulated upon neuronal differentiation., Materials and Methods: We observed the phenotype of olfactory stromal cells under DMEM/F12 plus 10% fetal bovine serum in comparison to cells from spheres induced by serum-free medium plus growth factors inducers of neural progenitors., Results: The expression of mesenchymal stromal (CD29+, CD73+, CD90+, CD45-), horizontal basal (ICAM-1/CD54+, p63+, p75NGFr+), and ensheathing progenitor cell (nestin+, GFAP+) proteins was determined in the cultured population by flow cytometry. The determination of Oct 3/4, Sox-2, and Mash-1 transcription factors, as well as the neurotrophins BDNF, NT3, and NT4 by RT-PCR in cells, was indicative of functional heterogeneity of the olfactory mucosa tissue sample., Conclusions: Mesenchymal and olfactory precursor proteins were downregulated by serum-free medium and promoted differentiation of mesenchymal stromal cells into neurons and astroglial cells.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are being investigated for their potential in the treatment of musculoskeletal injuries, including tendon and ligament lesions, and cartilage lesions. Culture expansion of cells has traditionally been performed in medium supplemented with fetal bovine serum (FBS), however, concerns regarding the antigenicity and potential viral or prion contamination of FBS have prompted interest in alternative medium supplements. Platelet lysate (PL) contains elevated concentrations of growth factors, including transforming growth factor-β (TGF-β), platelet-derived growth factors, and fibroblast growth factor, released from the α-granules of platelets; therefore, PL could be an ideal medium supplement. The effect of PL on mesenchymal stromal cell (MSC) growth and differentiation has not been fully elucidated. We hypothesized that PL medium would contain significantly higher amounts of TGF-β1 than FBS medium and would be associated with enhanced osteogenic and chondrogenic differentiation. MSCs were isolated from bone marrow collected from five adult horses. Cells were cultured in traditional medium supplemented with FBS or in medium supplemented with fibrinogen depleted-PL (FD-PL). Immunophenotyping was performed using flow cytometry. Trilineage differentiation was assessed through histology and gene expression analysis using quantitative reverse transcription-polymerase chain reaction. TGF-β1 was quantified in both medium types. The immunophenotypes of BM-MSCs cultured in FBS and FD-PL medium were similar with both culture types containing cells positive for stromal cell markers [cluster of differentiation 29 (CD29), CD44, CD90, CD105, and major histocompatibility complex I (MHCI)] and negative for exclusion markers (CD45, CD79α, and MHCII). Despite significantly higher TGF-β1 concentration in FD-PL medium, chondrogenic and osteogenic differentiation were not significantly different between FBS and FD-PL supplemented cultures. PL is an appropriate alternative medium supplement for the culture of equine BM-MSCs up to passage 3. However, despite increased TGF-β1 concentration in FD-PL medium, significant changes in chondrogenic differentiation compared with FBS medium should not be expected.

Mouse bone marrow-derived mast cells (mBMMCs) are an invaluable tool for the study of mast cell function as they represent a primary source of mature mast cells. They can be sourced from wild-type, knockout, and transgenic mice and are used to repopulate mast cell-deficient mice. This method describes the isolation of mast cell hematopoietic progenitors from the bone marrow of mouse femurs and their subsequent culture in an IL-3-rich culture medium. After 4 weeks in culture, mBMMCs are obtained in high number and are of high purity. Assessment of their granularity by toluidine staining and IgE receptor expression by flow cytometry is also described. These cells are a useful tool in the determination of in vitro and in vivo mast cell function in innate and adaptive immunity.

Fetal bovine serum (FBS) is widely used to culture mesenchymal stem cells (MSCs) in the laboratory; however, FBS has been linked to adverse immune-mediated reactions prompting the search for alternative cell culture medium. Platelet lysate (PL) as an FBS substitute has been shown to promote MSCs growth without compromising their functionality. Fibrinogen contained in PL has been shown to negatively impact the immune modulating properties of MSCs; therefore, we sought to deplete fibrinogen from PL and compare proliferation, viability, and immunomodulatory capacities of MSCs in FBS or PL without fibrinogen. We depleted fibrinogen from equine platelet lysate (ePL) and measured platelet-derived growth factor-beta (PDGF-β), transforming growth factor-beta (TGF-β) and tumor necrosis factor-alpha (TNF-α) through ELISA. First, we determined the ability of 10% ePL or fibrinogen-depleted lysate (fdePL) compared with 10% FBS to suppress monocyte activation by measuring TNF-α from culture supernatants. We then evaluated proliferation, viability, and immunomodulatory characteristics of bone marrow-derived MSCs (BM-MSCs) cultured in FBS or ePL with or without fibrinogen. Growth factor concentrations decreased in ePL after fibrinogen depletion. Lipopolysaccharide (LPS)-stimulated monocytes exposed to ePL and fdePL produced less TNF-α than LPS-stimulated monocytes in 10% FBS. BM-MSCs cultured in fdePL exhibited lower proliferation rates, but similar viability compared with BM-MSCs in ePL. BM-MSCs in fdePL did not effectively suppress TNF-α expression from LPS-stimulated monocytes compared with BM-MSCs in FBS. Depleting fibrinogen results in a lysate that suppresses TNF-α expression from LPS-stimulated monocytes, but that does not support proliferation and immune-modulatory capacity of BM-MSCs as effectively as nondepleted lysate.

The present study proposed to compare the impact of agitation mode (static, orbital, and mechanical) on the culture of mesenchymal stem cells extracted from the Wharton's jelly of umbilical cords (WJ-MSC), in a clinical grade culture medium, using human platelet lysate and different xeno-free microcarriers. Attachment, expansion, and detachment performances were characterized by a new dedicated tool of microscopic image posttreatment, allowing an in situ cell counting without detachment step. Results showed that performances in static mode were not necessarily representative of those obtained in dynamic mode. Moreover, impacts on nutrient consumptions and metabolite productions were identified, such as a higher glutamine consumption when Cytodex-1 microcarriers were used. The detachment strategy used was relatively efficient for Star-Plus, Plastic-Plus, and Hillex II, but not sufficient for Cytodex-1. Despite Cytodex-1 presented promising attachment and expansion performances, Star-Plus and Plastic-Plus showed a better compromise, respectively, for the orbital and the mechanical agitation modes., (© 2019 American Institute of Chemical Engineers.)

Cell-based therapies are increasingly focused on allogeneic stem cell sources because of several advantages in eliminating donor variability (e.g., aging and disease pathophysiology) affecting stem cell quality and in cell-banked sourcing of healthy donors to enable "off-the-shelf" products. However, allogeneic cell therapy is limited by host patient immunologic competence and inconsistent performance due to cell delivery methods. To address allogeneic cell therapy limitations, this study developed a new allogeneic stem cell sheet using human umbilical cord mesenchymal stem cells (hUC-MSC) that present low antigenicity (i.e., major histocompatibility complex, MHC). Optimal conditions including cell density, passage number, and culture time were examined to fabricate reliable hUC-MSC sheets. MHC II antigens correlated to alloimmune rejection were barely expressed in hUC-MSC sheets compared to other comparator MSC sheets (hBMSC and hADSC). hUC-MSC sheets easily graft spontaneously onto subcutaneous tissue in immune-deficient mice within 10 minutes of placement. No sutures are required to secure sheets to tissue because sheet extracellular matrix (ECM) actively facilitates cell-target tissue adhesion. At 10 days post-transplantation, hUC-MSC sheets remain on ectopic target tissue sites and exhibit new blood vessel formation. Furthermore, implanted hUC-MSC sheets secrete human HGF continuously to the murine target tissue. hUC-MSC sheets described here should provide new insights for improving allogenic cell-based therapies.

Background: Cell therapy has been proposed for patients with critical limb ischemia (CLI). Autologous bone marrow derived cells (BMCs) have been mostly used, mesenchymal stem cells (MSCs) being an alternative. The aim of this study was to characterize two types of MSCs and evaluate their efficacy., Methods: MSCs were obtained from CLI-patients BMCs. Stimulated- (S-) MSCs were cultured in endothelial growth medium. Cells were characterized by the expression of cell surface markers, the relative expression of 6 genes, the secretion of 10 cytokines and the ability to form vessel-like structures. The cell proangiogenic properties was analysed in vivo, in a hindlimb ischemia model. Perfusion of lower limbs and functional tests were assessed for 28 days after cell infusion. Muscle histological analysis (neoangiogenesis, arteriogenesis and muscle repair) was performed., Results: S-MSCs can be obtained from CLI-patients BMCs. They do not express endothelial specific markers but can be distinguished from MSCs by their secretome. S-MSCs have the ability to form tube-like structures and, in vivo, to induce blood flow recovery. No amputation was observed in S-MSCs treated mice. Functional tests showed improvement in treated groups with a superiority of MSCs and S-MSCs. In muscles, CD31+ and αSMA+ labelling were the highest in S-MSCs treated mice. S-MSCs induced the highest muscle repair., Conclusions: S-MSCs exert angiogenic potential probably mediated by a paracrine mechanism. Their administration is associated with flow recovery, limb salvage and muscle repair. The secretome from S-MSCs or secretome-derived products may have a strong potential in vessel regeneration and muscle repair. Trial registration NCT00533104.

Mesenchymal stem cells are an ideal source for regenerative medicine. For clinical use, cell culture should be done at stable conditions, thus the use of serum should be avoided because of the batch-to-batch variations of serum. Although several kinds of serum-free media are available, a method to confirm whether they contain serum has not been established yet. During studies on effect of adipocyte mesenchymal stem cells (Ad-MSCs) on pain using a human pain gene array, we noticed that BDKRB1 gene was constantly upregulated when serum was used in the culture medium. In this study, we attempted to establish further the potential of this gene as a new marker indicative of the presence of serum in media. Using a real-time quantitative PCR gene array screening containing 84 functional genes, we verified BDKRB1 as a specific gene upregulated in the presence of serum. The expression of BDKRB1 in Ad-MSCs was induced not only by bovine serum but also by human serum. The BDKRB1 expression was induced even when Ad-MSCs was cultured with 0.1% serum in the medium. We concluded that BDKRB1 is a valuable marker to detect traces of both human and animal serum in Ad-MSCs cultures. Our study provides a new method to confirm the absence of serum in media and ensure a stable cell culture condition., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Mineralization capability is an important issue in developing bone repairing biomaterials, while it is not quite clear how this feature would act in the presence of cells and influence cell osteogenic differentiation without adding extra osteoinductive factors such as β‑sodium glycerophosphate and dexamethasone. Poly(l‑lactide) (PLLA) and gelatin composite fibers (PG, 1:1 in weight) were electrospun, treated with CaCl 2 solution (PG-Ca), and used for mineralization studies by using cell culture media (αMEM, and αMEM + serum). Bone mesenchymal stromal cells (BMSCs) were then seeded and cultured on both PG and PG-Ca fibrous mats for 28 days by only using αMEM + serum. Interestingly, mineral depositions on both PG and PG-Ca fibers were detected in the environment of αMEM or αMEM + serum, in which, PG-Ca fibers demonstrated stronger ability in inducing hydroxyapatite formation than PG fibers, especially in the presence of fetal bovine serum. When BMSCs were cultured on the two kinds of fibrous mats, apatite depositions were still clearly detected, while the depositing amounts decreased in comparison with corresponding cell-free cases. It was ascribed to the consumption of ions by the continuously proliferating BMSCs, whose osteogenic differentiation was significantly promoted even without extra osteoinductive factors, especially on PG-Ca fibrous mats, in comparison with the control group. Therefore, it was confirmed the capability of scaffolding materials in enriching ions like calcium and phosphate around cells was an efficient way to promote bone regeneration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Pooled human platelet lysate (HPL) can replace fetal bovine serum (FBS) as xeno-free supplement for ex vivo expansion of mesenchymal stromal cells (MSCs). We evaluate here whether a double-virally-inactivated HPL (DVI-HPL) prepared from expired Intercept-treated platelet concentrates (PCs) and treated by solvent/detergent (S/D) can be used for MSC expansion., Study Design and Methods: Expired Intercept-treated PCs in 65% platelet (PLT) additive solution were pooled and subjected to a 1% tri-n-butyl phosphate/1% Triton X-45 treatment followed by soybean oil, hydrophobic interaction chromatography purification, and sterile filtration. Bone marrow-derived MSCs (BM-MSCs) were expanded for four passages in growth medium containing 10% DVI-HPL, I-HPL (from Intercept-PC only), untreated HPL, and FBS. MSC morphology, doubling time, immunophenotype, immunosuppressive activity, and differentiation capacity were compared., Results: Expanded cells had typical spindle morphology and showed higher viability in all HPL conditions than in FBS. The DVI-HPL and FBS-expanded cells were morphologically larger than in I-HPL and HPL supplements. The cumulative population doubling was lower using DVI-HPL than with HPL and I-HPL, but significantly higher than using FBS. Immunophenotype was not affected by the supplements used. Immunosuppressive activity was maintained with all supplements. Differentiation capacity into chondrocytes and osteocytes was more effective in DVI-HPL but less toward adipocytes compared to other supplements., Conclusions: Human PLT lysate made from Intercept-PCs subjected to S/D treatment may be an alternative to untreated HPL and to I-HPL for BM-MSC expansion. This finding reinforces the potential of HPL as a virally safe alternative to FBS for clinical grade MSC expansion protocols., (© 2019 AABB.)

After in vivo transplantation, mesenchymal stem cells (MSC) face an ischemic microenvironment, characterized by nutrient deprivation and reduced oxygen tension, which reduces their viability and thus their therapeutic potential. Therefore, MSC response to models of in vitro ischemia is of relevance for improving their survival and therapeutic efficacy. The aim of this study was to understand the survival/adaptive response mechanism that MSC use to respond to extreme culture conditions. Specifically, the effect of a long-term starvation on human bone marrow (hBM)-derived MSC cultured in a chemically defined medium (fetal bovine serum-free [SF] and human SF), either in hypoxic or normoxic conditions. We observed that hBM-MSC that were isolated and cultured in SF medium and subjected to a complete starvation for up to 75 days transiently changed their behavior and phenotype. However, at the end of that period, hBM-MSC retained their characteristics as determined by their morphology, DNA damage resistance, proliferation kinetic, and differentiation potential. This survival mode involved a quiescent state, confirmed by increased expression of cell cycle regulators p16, p27, and p57 and decreased expression of proliferating cell nuclear antigen (PCNA), Ki-67, mTOR, and Nanog. In addition, Jak/STAT (STAT6) antiapoptotic activity selected which cells conserved stemness and that supported metabolic, bioenergetic, and scavenging requirements. We also demonstrated that hBM-MSC exploited an autophagic process which induced lipid β-oxidation as an alternative energy source. Priming MSC by concomitant starvation and culture in hypoxic conditions to induce their quiescence would be of benefit to increase MSC survival when transplanted in vivo. Stem Cells 2019;37:813-827., (© AlphaMed Press 2019.)

As MSC products move from early development to clinical translation, culture conditions shift from xeno- to xeno-free systems. However, the impact of isolation and culture-expansion methods on the long-term resiliency of MSCs within challenging transplant environments is not fully understood. Recent work in our lab has shown that palmitate, a saturated fatty acid elevated in the serum of patients with obesity, causes MSCs to convert from an immunosuppressive to an immunostimulatory state at moderate to high physiological levels. This demonstrated that metabolically-diseased environments, like obesity, alter the immunomodulatory efficacy of healthy donor MSCs. In addition, it highlighted the need to test MSC efficacy not only in ideal conditions, but within challenging metabolic environments. To determine how the choice of xeno- vs. xeno-free media during isolation and expansion would affect future immunosuppressive function, umbilical cord explants from seven donors were subdivided and cultured within xeno- (fetal bovine serum, FBS) or xeno-free (human platelet lysate, PLT) medias, creating 14 distinct MSC preparations. After isolation and primary expansion, umbilical cord MSCs (ucMSC) were evaluated according to the ISCT minimal criteria for MSCs. Following baseline characterization, ucMSC were exposed to physiological doses of palmitate and analyzed for metabolic health, apoptotic induction, and immunomodulatory potency in co-cultures with stimulated human peripheral blood mononuclear cells. The paired experimental design (each ucMSC donor grown in two distinct culture environments) allowed us to delineate the contribution of inherent (nature) vs. environmentally-driven (nurture) donor characteristics to the phenotypic response of ucMSC during palmitate exposure. Culturing MSCs in PLT-media led to more consistent growth characteristics during the isolation and expansion for all donors, resulting in faster doubling times and higher cell yields compared to FBS. Upon palmitate challenge, PLT-ucMSCs showed a higher susceptibility to palmitate-induced metabolic disturbance, but less susceptibility to palmitate-induced apoptosis. Most striking however, was that the PLT-ucMSCs resisted the conversion to an immunostimulatory phenotype better than their FBS counterparts. Interestingly, examining MSC suppression of PBMC proliferation at physiologic doses of palmitate magnified the differences between donors, highlighting the utility of evaluating MSC products in stress-based assays that reflect the challenges MSCs may encounter post-transplantation.

Mesenchymal stem cells (MSCs) have been demonstrated to be involved in tumor progression and the modulation of the tumor microenvironment, partly through their secretome. Extracellular vesicles (EVs) are membranous nanovesicles secreted by multiple types of cells and have been demonstrated to mediate intercellular communication in both physiological and pathological conditions. However, numerous questions still remain regarding the underlying mechanisms and functional consequences of these interactions. The purpose of this study was to investigate the effects of human umbilical cord mesenchymal stem cell‑derived EVs (hUC‑MSC‑EVs) on the proliferation, migration and invasion of human breast cancer cells. We successfully generated and identified hUC‑MSCs and hUC‑MSC‑EVs which were used in this study. The results revealed that treatment of the MDA‑MB‑231 and MCF‑7 human breast cancer cells with medium containing hUC‑MSC‑EVs significantly enhanced the proliferation, migration and invasion of the cells in vitro. Treatment of the cells with medium containing hUC‑MSC‑EVs also reduced E‑cadherin expression and increased N‑cadherin expression, thus promoting the epithelial‑mesenchymal transition (EMT) of the breast cancer cells. Treatment of the breast cancer cells with extracellular signal‑regulated kinase (ERK) inhibitor prior to the interaction with hUC‑MSC‑EVs significantly reversed the enhanced proliferation, migration and invasion, as well as the EMT of the breast cancer cells induced by the hUC‑MSC‑EVs. On the whole, these data indicate that hUC‑MSC‑EVs promote the invasive and migratory potential of breast cancer cells through the induction of EMT via the ERK pathway, leading to malignant tumor progression and metastasis. Taken together, the findings of this study suggest that targeting pathways to reverse EMT may lead to the development of novel therapeutic approaches with which to combat breast cancer.

The objective of the present study was to evaluate the effect of porcine Mesenchymal Stem Cells (MSCs) secreted factors on bovine in vitro embryo development by using MSCs in different culture systems: SOF medium, SOF medium conditioned by MSCs in monolayer and in reverse drop and by embryo culture in co-culture with MSCs. Statistically highly significant differences were noted between the number of blastocysts derived cultures in all tested culture systems. The in vitro culture in SOF turned out to be the most optimal. Statistically highly significant differences were observed in the number of blastocyst obtained between SOF and SOF in co-culture with MSCs (p < 0.0001), and between SOF and SOF conditioned (monolayer and drop) (p < 0.00001). The trials to produce blastocysts in SOF conditioned by MSCs in reverse drops and monolayer failed. The blastocysts were obtained and analysed by TUNEL only in two out of four experimental groups: SOF and SOF in co-culture with MSCs. There were no significant differences between any of analysed blastocysts' groups neither in the total number of nuclei nor in the apoptotic features. Neither medium conditioning by MSCs in monolayer and in reverse drop nor embryo culture in co-culture with MSC turned out to be effective.

Adipose-derived stem cells (ASCs) are multipotent and immune-privileged mesenchymal cells, making them ideal candidates for therapeutic purposes to manage tendon disorders. Providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices. To this aim we investigated the in vitro tenogenic differentiation potential of ASCs using a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL) suitable for cell therapy and both supplemented with CTGF, TGFβ-3, BMP-12 and ascorbic acid (AA) soluble factors. Human ASCs were isolated from 4 healthy donors and they were inducted to differentiate until 14 days in both hPL and SF tenogenic media (hPL-TENO and SF-TENO). Cell viability and immunophenotype profile were analysed to evaluate mesenchymal stem cell (MSC) characteristics in both xenogenic-free media. Moreover, the expression of stemness and tendon-related markers upon cell differentiation by RT-PCR, protein staining and cytofluorimetric analysis were also performed. Our results showed the two xenogenic-free media well support cell viability of ASCs and maintain their MSC nature as demonstrated by their typical immunophenototype profile and by the expression of NANOG, OCT4 and Ki67 genes. Moreover, both hPL-TENO and SF-TENO expressed significant high levels of the tendon-related genes SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 already at early time points in comparison to the respective controls. Significant up-regulations in scleraxis, collagen and tenomodulin proteins were also demonstrated at in both differentiated SF and hPL ASCs. In conclusion, we demonstrated firstly the feasibility of both serum and xenogenic-free media tested to culture ASCs moving forward the GMP-compliant approaches for clinical scale expansion of human MSCs needed for therapeutical application of stem cells. Moreover, a combination of CTGF, BMP-12, TGFβ3 and AA factors strongly and rapidly induce human ASCs to differentiate into tenocyte-like cells., Competing Interests: The authors have declared that no competing interests exist.

In order to investigate the influence of calcium and strontium ion concentration on human bone marrow stromal cells and their differentiation to osteoblasts, different cell culture media have been used. Even though this study does not contain a bone substitute material, the reason for this study was the decrease of cation concentration by many biomaterials, due to induced apatite precipitation. As a consequence, the reduced calcium ion concentration is known to affect osteoblastic development. Therefore, the main focus was put on the question, whether an increased strontium concentration (in the range of mM) might be suitable to compensate the lack of calcium ions. The effect of solely strontium ions-with only calcium in the media resulting from fetal calf serum-was investigated. Commercially available calcium-free medium (modified α-MEM) was tested in comparison with media with varied calcium ion concentrations (0.9, 1.8, and 3.6 mM), or strontium ion concentration (0.4, 0.9, 1.8, and 3.6 mM). In case of calcium, higher concentrations cause increased proliferation, while differentiation was shifted to earlier points of time. Differentiation was increased by solely strontium ions only at 0.4-0.9 mM, while proliferation was highest for 0.9-1.8 mM. From these results, it can be concluded that strontium is able to compensate a lack of calcium to a certain degree. Thus, in contrast to calcium ion release, a strontium ion release from bone substitute materials might be applicable for stimulation of bone regeneration without influencing the media saturation., (© 2018 Japanese Society of Developmental Biologists.)

Neural regeneration is of great interest due to its potential to treat traumatic brain injuries and diseases that impact quality of life. Growth factor mediated differentiation can take up to several weeks to months to produce the cell of interest whereas chemical stimulation may be as minimal as a few hours. The smaller time scale is of great clinical relevance. Adipose derived stem cells (ADSCs) were treated for up to 24 h with a novel differentiation media containing the cyclic ketamine compounds to direct neurogenic induction. The extent of differentiation was investigated by proteome changes occurring during the process. The treatments indicated the ADSCs responded favorably to the neurogenic induction media by presenting a number of morphological cues of neuronal phenotype previously seen and a higher cell population post induction compared to previous studies. Furthermore, approximately 3500 proteins were analyzed and identified by mass spectrometric iTRAQ analyses. The bioinformatics analyses revealed hundreds of proteins whose expression level changes were statistically significant and biologically relevant to neurogenesis and annotated as being involved in neurogenic development. Complementing this, the Bioplex cytokine assay profiles present evidence of decreased panel of stress response cytokines and a relative increase in those involved in neurogenesis.

Human mesenchymal stem cells (MSC) are being explored for cell therapies targeting varied human diseases. For that, cells are being expanded in vitro, many times with fetal bovine serum (FBS) as the main source of growth factors. However, animal-derived components should not be used, to avoid immune rejection from the patient that receives the MSC. To solve this issue, different xeno-free media are being developed, and an industrial-grade human plasma fraction (SCC) is a promising candidate to substitute FBS. Indeed, we have previously shown that MSC expanded in SCC-medium maintain their phenotype and genetic stability. However, a reduction on MSC motility was observed when comparing with MSC motility on FBS-medium. Thus, in this present study, we have tested different factors to improve the motility of MSC in SCC-medium. Time lapse assays and experiments with transwells revealed that supplementation of the xeno-free medium with FGF or PDGF, but not TNF-α or SDF-1, increased MSC motility. Interestingly, FGF and PDGF supplementation also led to alterations on MSC morphology to a shape similar to the one observed when using FBS. The mechanism behind the effect of FGF on MSC motility involved the increased expression of αVβ3 integrin. Furthermore, assays with small molecule inhibitors revealed that the signalling molecule p38 MAPK is important for MSC motility and that MEK/ERK and PI3K/AKT also have a role on FGF-supplemented expanded MSC. Thus, it was found that FGF supplementation can improve the motility of xeno-free-expanded MSC and that the cells motility is regulated by αVβ3 integrin., (© 2018 John Wiley & Sons, Ltd.)

This study aimed to evaluate the use of Insulin-Transferrin-Selenium (ITS) medium in place of fetal bovine serum (FBS) to culture human amnion mesenchymal stem cells (hAMSCs). Cell morphology, ultrastructure, proliferation, migration and MSC related markers were assessed accordingly. The hAMSCs were induced to osteocyte, chondrocyte, adipocyte and keratinocyte by culturing in appropriate induction medium. hAMSCs mRNA expression was detected for the matrix metalloproteinases 2 (MMP2), keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-I), Platelet-derived Growth Factor (PDGF), and transforming growth factor beta 1 (TGF-β) by real-time quantitative RT-PCR. Our results showed that hAMSCs cultured in ITS medium exhibited similar proliferation rates, demonstrated a statistically significant increased migration and expressed similar levels of MSC markers(CD73+, CD90+, CD105+, CD45-, CD34-) compared with those cultured in FBS. Osteoblasts, chondrocytes, adipocytes and keratinocytes were differentiated. Results of transmission electron microscope (TEM) revealed that hAMSCs cultured in ITS medium underwent active metabolism. The mRNA expression of MMP2, VEGF, KGF, TGF-β, IGF-I and PDGF upregulated in ITS medium. In conclusion, ITS medium has the potential to be used for the expansion of hAMSCs before clinical application., (© 2019 by the Association of Clinical Scientists, Inc.)