Ecografía abdominal básica en medicina intensiva y VExUS Score / Abdominal ultrasound and VExUS score in critical care

El uso de la ecografía en las unidades de críticos se ha extendido de forma exponencial en las últimas dos décadas y se ha convertido en una parte esencial de nuestra práctica clínica. La ecografía abdominal es una técnica ampliamente establecida en otras especialidades, pero su uso en cuidados intensivos ha quedado rezagado respecto a otras modalidades de ecografía. Sin embargo, su potencial papel en el diagnóstico y manejo de los pacientes lo convertirá en una herramienta invaluable para los intensivistas. El uso más extendido de la ecografía abdominal a pie de cama es para la valoración de la presencia de líquido libre en el paciente traumático. No obstante, la ecografía abdominal también puede ayudarnos a diagnosticar pacientes con dolor abdominal, hipovolemia o anuria, y puede guiarnos en procedimientos como la paracentesis o el sondaje vesical o gástrico. (AU)

The use of ultrasound while caring for critically ill patients has been increasing exponentially in the last two decades and now is an essential component of intensive care practice. Abdominal ultrasound is an established technique in other specialties, but its use in intensive care has lagged behind other ultrasound modalities. However, its potential role in the diagnosis and management of patients will make it an invaluable tool for intensivists. The main use of abdominal ultrasound at the bedside is for free fluid detection in trauma patients. But abdominal ultrasound can also help us diagnose patients with abdominal pain, hypovolemia or anuria, and it can guide us during procedures such as paracentesis or bladder catheter and gastric tube placement. (AU)

Induced pluripotent stem cells in companion animals: how can we move the field forward?

Following a one medicine approach, the development of regenerative therapies for human patients leads to innovative treatments for animals, while pre-clinical studies on animals provide knowledge to advance human medicine. Among many different biological products under investigation, stem cells are among the most prominent. Mesenchymal stromal cells (MSCs) are extensively investigated, but they present challenges such as senescence and limited differentiation ability. Embryonic stem cells (ESCs) are pluripotent cells with a virtually unlimited capacity for self-renewal and differentiation, but the use of embryos carries ethical concerns. Induced pluripotent stem cells (iPSCs) can overcome all of these limitations, as they closely resemble ESCs but are derived from adult cells by reprogramming in the laboratory using pluripotency-associated transcription factors. iPSCs hold great potential for applications in therapy, disease modeling, drug screening, and even species preservation strategies. However, iPSC technology is less developed in veterinary species compared to human. This review attempts to address the specific challenges associated with generating and applying iPSCs from companion animals. Firstly, we discuss strategies for the preparation of iPSCs in veterinary species and secondly, we address the potential for different applications of iPSCs in companion animals. Our aim is to provide an overview on the state of the art of iPSCs in companion animals, focusing on equine, canine, and feline species, as well as to identify which aspects need further optimization and, where possible, to provide guidance on future advancements. Following a "step-by-step" approach, we cover the generation of iPSCs in companion animals from the selection of somatic cells and the reprogramming strategies, to the expansion and characterization of iPSCs. Subsequently, we revise the current applications of iPSCs in companion animals, identify the main hurdles, and propose future paths to move the field forward. Transferring the knowledge gained from human iPSCs can increase our understanding in the biology of pluripotent cells in animals, but it is critical to further investigate the differences among species to develop specific approaches for animal iPSCs. This is key for significantly advancing iPSC application in veterinary medicine, which at the same time will also allow gaining pre-clinical knowledge transferable to human medicine.

Programa de Mentoría: acompañando tus primeros pasos hacia la excelencia científica / Mentoring Programme: Guiding your first steps towards scientific excellence

El primer Programa de Mentoría de SEMICYUC tiene como objetivo apoyar la carrera investigadora de los miembros más jóvenes de la Sociedad. Como beneficios añadidos está la adquisición de nuevas capacidades de investigación y/o clínicas, incrementar la capacidad de reflexión y fomentar el desarrollo de la próxima generación de líderes en la investigación. Este proyecto no sería posible sin el equipo excepcional de mentores o expertos investigadores dispuestos a emprender el viaje con los jóvenes aprendices. El presente artículo expone las bases de dicho programa, además de proponer futuros cambios en haz de una mejora continua (AU)

SEMICYUC's first Mentoring Programme aims to support the research careers of the Society's youngest members. Added benefits include acquiring new research and/or clinical skills, increasing the ability of critical thought, and fostering the development of the next generation of research leaders. This project would not be possible without the exceptional team of mentors or research experts willing to embark on the journey with the young trainees. This article sets out the foundations of such a programme and proposes future changes for continuous improvement (AU)

Laparoscopically Assisted Percutaneous Inguinal Ring Closure for Resolution of Inguinal/Scrotal Hernias in Rams: Cadaveric Study and Three Cases Report.

The aim of this study is to evaluate a laparoscopically assisted percutaneous suture (LAPS) procedure to treat inguinal hernia (IH) while preserving testicles in rams. An ex vivo experiment with six ram cadavers and a report of three clinical cases are discussed. In cadavers, both internal inguinal rings (IIR) were partially closed by LAPS. Two LAPS methods were tested: (1) using a laparoscopic portal closure device and (2) using a suture loop inserted through needles in each IIR. After each procedure, the closure was laparoscopically evaluated and the number of U- sutures was recorded. The procedure was also performed on three client-owned rams with unilateral non-strangulated IH and the occurrence of re-herniation was followed up. In cadavers, LAPS of the IIRs could be easily and satisfactorily performed with either of the two systems, requiring one to three U-sutures per IIR. No differences were observed between the two surgical procedures. In two clinical cases, the procedure was successfully performed without reoccurrence of herniation or alterations in reproductive behavior in the following 3 and 6 months. In the third case, the hernia was reduced but a retroperitoneal emphysema during laparoscopy prevented hernioplasty and the animal herniated again. In conclusion, LAPS of IIR can be used as a simple and feasible treatment to preserve testicles in rams with IH.

Mentoring Programme: Guiding your first steps towards scientific excellence.

SEMICYUC's first Mentoring Programme aims to support the research careers of the Society's youngest members. Added benefits include acquiring new research and/or clinical skills, increasing the ability of critical thought, and fostering the development of the next generation of research leaders. This project would not be possible without the exceptional team of mentors or research experts willing to embark on the journey with the young trainees. This article sets out the foundations of such a programme and proposes future changes for continuous improvement.

Development of a laparoscopic technique for inguinal hernioplasty in standing horses.

BACKGROUND: Most previously described techniques for laparoscopic inguinal hernioplasty (IH) in horses require advanced laparoscopic skills. Our objective was to describe a new laparoscopic IH technique using a surgical anchoring system. METHODS: Standing laparoscopic IH was performed unilaterally in eight experimental stallions, using the contralateral inguinal canal (IC) as a control. A polyether ether ketone harpoon was anchored in the craniolateral aspect of the vaginal ring, and an extracorporeal knot was used to fix the device. Clinical evaluation, including testicular palpation and lameness examination, was conducted before and for 4 weeks after surgery. Repeat laparoscopy was performed 28 days later. RESULTS: Standing laparoscopic IH was performed in all horses with a surgical time of 38 ± 12.85 minutes. In two animals, a small peritoneal tear occurred that did not require repair. No other complications were recorded. On repeat laparoscopy, all devices were in place, and the IC remained partially closed in all horses. LIMITATIONS: The procedure was performed on normal experimental horses and has not been employed on horses that have had an inguinal hernia. CONCLUSIONS: This new standing laparoscopic hernioplasty technique provides another potential method for simple partial closure of the IC in stallions at risk of or with history of inguinal herniation.

Complications in Laparoscopic Access in Standing Horses Using Cannula and Trocar Units Developed for Human Medicine.

First cannulation is a critical manoeuvre in equine laparoscopy. This retrospective study aimed at the comparison of the frequency and type of complications detected when using different human laparoscopy devices for laparoscopic access in standing horses, and the influence of body condition in such complications. Forty-four procedures were included, and retrieved data comprised cannula insertion technique, body condition, and type and frequency of complications. Laparoscopic access techniques were classified into five groups: P: pneumoperitoneum created using Veress needle prior to cannulation; T: sharp trocar; D: direct access via surgical incision; V: Visiport optical trocar and H: optical helical cannula (OHC). In groups T, D, V and H, access was achieved without prior induction of pneumoperitoneum. Complications were registered in 13/44 procedures, of which retroperitoneal insufflation was the most common (6/13). Statistically significant association was found between the complication incidence and the type of access, with group D showing the highest complication frequency (80%) and group H the lowest frequency (0%). The majority of complications (9/13) were observed in overweight horses. We conclude that devices designed for human patients can be used for laparoscopic access in standing horses, with the use of OHC minimizing the appearance of complications, especially in overweight horses with OW.

Abdominal ultrasound and VExUS score in critical care.

The use of ultrasound while caring for critically ill patients has been increasing exponentially in the last two decades and now is an essential component of intensive care practice. Abdominal ultrasound is an established technique in other specialties, but its use in intensive care has lagged behind other ultrasound modalities. However, its potential role in the diagnosis and management of patients will make it an invaluable tool for intensivists. The main use of abdominal ultrasound at the bedside is for free fluid detection in trauma patients. But abdominal ultrasound can also help us diagnose patients with abdominal pain, hypovolemia or anuria, and it can guide us during procedures such as paracentesis or bladder catheter and gastric tube placement.

The immunomodulation-immunogenicity balance of equine Mesenchymal Stem Cells (MSCs) is differentially affected by the immune cell response depending on inflammatory licensing and major histocompatibility complex (MHC) compatibility.

The immunomodulatory properties of equine mesenchymal stem cells (MSCs) are important for their therapeutic potential and for their facilitating role in their escape from immune recognition, which may also be influenced by donor-recipient major histocompatibility complex (MHC) matching/mismatching and MHC expression level. Factors such as inflammation can modify the balance between regulatory and immunogenic profiles of equine MSCs, but little is known about how the exposure to the immune system can affect these properties in equine MSCs. In this study, we analyzed the gene expression and secretion of molecules related to the immunomodulation and immunogenicity of equine MSCs, either non-manipulated (MSC-naive) or stimulated by pro-inflammatory cytokines (MSC-primed), before and after their exposure to autologous or allogeneic MHC-matched/-mismatched lymphocytes, either activated or resting. Cytokine priming induced the immunomodulatory profile of MSCs at the baseline (MSCs cultured alone), and the exposure to activated lymphocytes further increased the expression of interleukin 6 (IL6), cyclooxygenase 2, and inducible nitric oxide synthase, and IL6 secretion. Activated lymphocytes were also able to upregulate the regulatory profile of MSC-naive to levels comparable to cytokine priming. On the contrary, resting lymphocytes did not upregulate the immunomodulatory profile of equine MSCs, but interestingly, MSC-primed exposed to MHC-mismatched lymphocytes showed the highest expression and secretion of these mediators, which may be potentially linked to the activation of lymphocytes upon recognition of foreign MHC molecules. Cytokine priming alone did not upregulate the immunogenic genes, but MSC-primed exposed to activated or resting lymphocytes increased their MHC-I and MHC-II expression, regardless of the MHC-compatibility. The upregulation of immunogenic markers including CD40 in the MHC-mismatched co-culture might have activated lymphocytes, which, at the same time, could have promoted the immune regulatory profile aforementioned. In conclusion, activated lymphocytes are able to induce the equine MSC regulatory profile, and their effects seem to be additive to the priming action. Importantly, our results suggest that the lymphocyte response against MHC-mismatched MSC-primed would promote further activation of their immunomodulatory ability, which eventually might help them evade this reaction. Further studies are needed to clarify how these findings might have clinical implications in vivo, which will help developing safer and more effective therapies.

Equine Mesenchymal Stem Cells Influence the Proliferative Response of Lymphocytes: Effect of Inflammation, Differentiation and MHC-Compatibility.

Immunomodulation and immunogenicity are pivotal aspects for the therapeutic use of mesenchymal stem cells (MSCs). Since the horse is highly valuable as both a patient and translational model, further knowledge on equine MSC immune properties is required. This study analysed how inflammation, chondrogenic differentiation and compatibility for the major histocompatibility complex (MHC) influence the MSC immunomodulatory-immunogenicity balance. Equine MSCs in basal conditions, pro-inflammatory primed (MSC-primed) or chondrogenically differentiated (MSC-chondro) were co-cultured with either autologous or allogeneic MHC-matched/mismatched lymphocytes in immune-suppressive assays (immunomodulation) and in modified one-way mixed leukocyte reactions (immunogenicity). After co-culture, frequency and proliferation of T cell subsets and B cells were assessed by flow cytometry and interferon-É£ (IFNÉ£) secretion by ELISA. MSC-primed showed higher regulatory potential by decreasing proliferation of cytotoxic and helper T cells and B cells. However, MHC-mismatched MSC-primed can also activate lymphocytes (proliferative response and IFNÉ£ secretion), likely due to increased MHC-expression. MSC-chondro maintained their regulatory ability and did not increase their immunogenicity, but showed less capacity than MSC-primed to induce regulatory T cells and further stimulated B cells. Subsequent in vivo studies are needed to elucidate the complex interactions between MSCs and the recipient immune system, which is critical to develop safe and effective therapies.

The Usefulness of Mesenchymal Stem Cells beyond the Musculoskeletal System in Horses.

The differentiation ability of mesenchymal stem cells (MSCs) initially raised interest for treating musculoskeletal injuries in horses, but MSC paracrine activity has widened their scope for inflammatory and immune-mediated pathologies in both equine and human medicine. Furthermore, the similar etiopathogenesis of some diseases in both species has advanced the concept of "One Medicine, One Health". This article reviews the current knowledge on the use of MSCs for equine pathologies beyond the locomotor system, highlighting the value of the horse as translational model. Ophthalmologic and reproductive disorders are among the most studied for MSC application. Equine asthma, equine metabolic syndrome, and endotoxemia have been less explored but offer an interesting scenario for human translation. The use of MSCs in wounds also provides a potential model for humans because of the healing particularities in both species. High-burden equine-specific pathologies such as laminitis have been suggested to benefit from MSC-therapy, and MSC application in challenging disorders such as neurologic conditions has been proposed. The available data are preliminary, however, and require further development to translate results into the clinic. Nevertheless, current evidence indicates a significant potential of equine MSCs to enlarge their range of application, with particular interest in pathologies analogous to human conditions.

Allo-antibody production after intraarticular administration of mesenchymal stem cells (MSCs) in an equine osteoarthritis model: effect of repeated administration, MSC inflammatory stimulation, and equine leukocyte antigen (ELA) compatibility.

BACKGROUND: Antibody production after allogeneic administration of mesenchymal stem cells (MSCs) could impact their clinical application. Proinflammatory priming of MSCs can potentiate their regulatory ability in vivo but increased expression of major histocompatibility complex (MHC) might augment their immunogenicity, potentially leading to immune memory thus limiting repeated allogeneic administration. This study aimed at evaluating the production of cytotoxic allo-antibodies directed against donor's ELA (equine leukocyte antigen) in mismatched and halfmatched horses receiving repeated intraarticular administration of stimulated MSCs (MSC-primed) and unstimulated MSCs (MSC-naïve) in pathologic joints. METHODS: From available stored samples from a previous in vivo study, cells from one donor and serially collected sera (five time-points) from three groups of recipients were used based on their ELA haplotypes to perform microcytotoxicity assays: Group 1 recipients mismatched with the donor that received MSC-naïve (naïve-mismatched recipients); Group 2 recipients mismatched with the donor that received MSC-primed (primed-mismatched recipients); Group 3 recipients halfmatched with the donor (sharing 1/2 haplotypes) that received MSC-primed (primed-halfmatched recipients). Sera from recipients (neat, 1:2 and 1:16 dilution) were tested against target cells from the donor (cryopreserved and expanded MSC-naïve and MSC-primed) or from one animal presenting the same ELA haplotypes than the donor (fresh peripheral blood lymphocytes as control). RESULTS: One to three weeks after first MSC administration, all recipient groups produced allo-antibodies regardless of MSC received (naïve or primed) and matching degree with donor. However, secondary response after MSC re-exposure was less evident in halfmatched recipients (MSC-primed) than in mismatched ones (both MSC-naïve and MSC-primed). Recipients of MSC-primed (both mismatched and halfmatched) tended towards developing lower antibody response than MSC-naïve recipients in vivo, but MSC-primed were targeted to death in higher percentage in vitro in the microcytoxicity assay. CONCLUSIONS: After first intraarticular allogeneic administration, the immunomodulatory profile of MSC-primed would have led to lower antibody production, but these antibodies would target more easily MSC-primed after second injection (re-exposure), likely because of their higher MHC expression.

Resident memory T cells are a cellular reservoir for HIV in the cervical mucosa.

HIV viral reservoirs are established very early during infection. Resident memory T cells (TRM) are present in tissues such as the lower female genital tract, but the contribution of this subset of cells to the pathogenesis and persistence of HIV remains unclear. Here, we show that cervical CD4+TRM display a unique repertoire of clusters of differentiation, with enrichment of several molecules associated with HIV infection susceptibility, longevity and self-renewing capacities. These protein profiles are enriched in a fraction of CD4+TRM expressing CD32. Cervical explant models show that CD4+TRM preferentially support HIV infection and harbor more viral DNA and protein than non-TRM. Importantly, cervical tissue from ART-suppressed HIV+ women contain high levels of viral DNA and RNA, being the TRM fraction the principal contributor. These results recognize the lower female genital tract as an HIV sanctuary and identify CD4+TRM as primary targets of HIV infection and viral persistence. Thus, strategies towards an HIV cure will need to consider TRM phenotypes, which are widely distributed in tissues.

Effect of allogeneic platelet lysate on equine bone marrow derived mesenchymal stem cell characteristics, including immunogenic and immunomodulatory gene expression profile.

Propagation ex vivo of mesenchymal stem cells (MSCs) requires culture medium supplementation. Fetal bovine serum (FBS) has long been the gold standard supplement, but its use is being questioned mainly due to ethical and safety issues. The use of platelet lysate (PL) as substitute of FBS has been proposed but little is known about its effects on equine MSCs characteristics including their immune profile. The aim of this work was to investigate for the first time the effect of allogenic PL on the immunogenic and immunomodulatory gene expression profile of equine bone marrow derived MSCs (eBM-MSCs) as well as on their proliferation ability, phenotype markers, and viability post-cryopreservation. The eBM-MSCs (n = 3) cultures were supplemented with 20% of allogeneic pooled concentrated PL (CPL; 591 × 103 platelets/µL) or basal PL (BPL; 177 × 103 platelets/µL) from three donors, using 10% FBS supplementation as control. The proliferative ability of eBM-MSCs under the three conditions was evaluated by calculating the cell doubling times (DT) up to passage 3 (P3) and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at P3. Viability of eBM-MSCs post-cryopreserved with CPL or FBS was assessed at 15, 30 and 60 days. The gene expression profile of eBM-MSCs was evaluated in P3 by RT-qPCR for characterization, immunogenic and immunomodulatory markers. The cells cultured in CPL had significantly higher ability to proliferate than with FBS or BPL (P < 0.001) in the MTT assay. Post-cryopreserved viability was similar between cells cultured and preserved in FBS and CPL at all time-points. Gene expression of MSC characterization markers was similar among the three conditions. The gene expression of the immunogenic markers MHC-I, MHC-II and CD40 was slightly (non-significant) increased in CPL condition compared to FBS and BPL. The CPL condition showed higher expression of the genes coding for the immunomodulatory molecules VCAM-1 (non-significant) and IL-6 (P < 0.05), and similar for COX-2; whereas iNOS and IDO were not expressed under any condition. In conclusion, the replacement of FBS by allogeneic CPL as a supplement for ex vivo propagation of eBM-MSCs provides appropriate proliferation and cryopreservation, and mildly upregulates the gene expression of immunomodulatory markers, thus constituting a potentially suitable alternative to the use of FBS. Further studies are needed to clarify the composition and effects of CPL supplementation on equine MSCs immunological profile.

Application of a laparoscopic technique for vasectomy in standing horses.

This report describes a technique for standing laparoscopic vasectomy in stallions through a prospective descriptive study. A preliminary study was carried out with two experimental intact male horses and subsequently the procedure was performed in two clinical cases. These horse owners want to keep their animals in the most possible natural way, preserving its stallion behaviour in a herd without generating offspring. The horses were sedated and restrained in stocks and laparoscopic vasectomy was performed using three portal sites in both paralumbar fossae recording surgical times. A 4-cm segment of each ductus deferens (DD) was occluded with laparoscopic vessel sealing devices and subsequently excised. Semen collection was performed using an artificial vagina before the laparoscopic procedure and at 15 and 60 days postoperatively. Sexual behaviour and spermiogram were analysed. Two months after vasectomy, control laparoscopy was performed in experimental horses to assess the surgical site. Bilateral vasectomy could be performed without intraoperative complications in a mean surgical time of 20 min per DD. Success of the procedure was confirmed in all cases by azoospermic ejaculates 60 days after vasectomy. This is the first time that the technique for laparoscopic vasectomy is described in horses.

Assessment of effectiveness and safety of repeat administration of proinflammatory primed allogeneic mesenchymal stem cells in an equine model of chemically induced osteoarthritis.

BACKGROUND: This study aimed at assessing the effectiveness and safety of repeated administrations of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) primed with tumor necrosis factor (TNF)-α and interferon-γ in an equine model of chemically-induced osteoarthritis. Arthritis was induced in both radio-carpal (RC)-joints by amphotericin-B in 18 ponies, divided into three groups depending on the treatment injected: MSC-naïve (n = 7), MSC-primed (n = 7) and control (n = 4). The study consisted of two phases and used one RC-joint of each animal in each phase, with four months time-lapse, in order to assess two end-points. Clinical, synovial, radiological and ultrasonographic follow-up was performed. At six months, animals were euthanized and both carpi were assessed by magnetic resonance imaging (MRI), gross anatomy, histopathology, histochemistry and gene expression. RESULTS: Clinical and synovial inflammatory signs were quicker reduced in MSC-treated groups and repeated allogeneic administration did not produce adverse reactions, but MSC-primed group showed slight and transient local inflammation after second injection. Radiology and MRI did not show significant differences between treated and control groups, whereas ultrasonography suggested reduced synovial effusion in MSC-treated groups. Both MSC-treated groups showed enhanced cartilage gross appearance at two compared to six months (MSC-naïve, p < 0.05). Cartilage histopathology did not reveal differences but histochemistry suggested delayed progression of proteoglycan loss in MSC-treated groups. Synovium histopathology indicated decreased inflammation (p < 0.01) in MSC-primed and MSC-naïve at two and six months, respectively. At two months, cartilage from MSC-primed group significantly (p < 0.05) upregulated collagen type II (COL2A1) and transforming growth factor (TGF)-ß1 and downregulated cyclooxygenase-2 and interleukin (IL)-1ß. At six months, MSC-treatments significantly downregulated TNFα (p < 0.05), plus MSC-primed upregulated (p < 0.05) COL2A1, aggrecan, cartilage oligomeric protein, tissue inhibitor of metalloproteinases-2 and TGF-ß1. In synovium, both MSC-treatments decreased (p < 0.01) matrix metalloproteinase-13 expression at two months and MSC-primed also downregulated TNFα (p < 0.05) and IL-1ß (p < 0.01). CONCLUSIONS: Both MSC-treatments provided beneficial effects, mostly observed at short-term. Despite no huge differences between MSC-treatments, the findings suggested enhanced anti-inflammatory and regulatory potential of MSC-primed. While further research is needed to better understand these effects and clarify immunogenicity implications, these findings contribute to enlarge the knowledge about MSC therapeutics and how they could be influenced.

Differentiation of equine bone marrow derived mesenchymal stem cells increases the expression of immunogenic genes.

Mesenchymal stem cells (MSCs) are a promising treatment for equine musculoskeletal injuries because of their ability to regulate the inflammation and to differentiate into other cell types. Since interest in allogeneic therapy is rising, concerns about MSC immunogenicity need to be addressed. Differentiated MSCs from several species increase their expression of immunogenic molecules and induce alloresponses, but equine MSC immunogenic profile after differentiation has not been reported. Therefore, the aim of this study was to assess the gene expression of immunogenic markers in tri-lineage differentiated equine bone marrow derived MSCs (eBM-MSCs). For this purpose, eBM-MSCs (n = 4) were differentiated into osteoblasts, adipocytes and chondrocytes. Differentiation was confirmed by specific staining and gene expression of lineage-related markers. Subsequently, gene expression of MHC-I, MHC-II, CD40 and CD80 was analyzed in undifferentiated (control) and tri-lineage differentiated eBM-MSCs. Osteogenesis and adipogenesis, but not chondrogenesis, significantly upregulated MHC-I; MHC-II expression significantly increased in the three lineages, while CD40 and CD80 expression did not change. Despite this, MHC-I and MHC-II upregulation after differentiation might lead to increased immunogenicity and risk of allorecognition, either eBM-MSCs differentiate in vivo after administration or they are differentiated prior to administration, with potential negative consequences for effectiveness and safety of allogeneic therapy.