The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

By employing multiparameter sorting, we identified in murine bone marrow (BM) a homogenous population of rare (B0.02% of BMMNC) Sca-1 þ lin À CD45 À cells that express by RQ-PCR and immunohistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1. The direct electronmicroscopical analysis revealed that these cells are small (B2-4 lm), posses large nuclei surrounded by a narrow rim of cytoplasm, and contain open-type chromatin (euchromatin) that is typical for embryonic stem cells. In vitro cultures these cells are able to differentiate into all three germ-layer lineages. The number of these cells is highest in BM from young (B1-monthold) mice and decreases with age. It is also significantly diminished in short living DBA/2J mice as compared to long living B6 animals. These cells in vitro respond strongly to SDF-1, HGF/SF and LIF and express CXCR4, c-met and LIF-R, respectively, and since they adhere to fibroblasts they may be coisolated with BM adherent cells. We hypothesize that this population of Sca-1 þ lin À CD45 À very small embryonic-like (VSEL) stem cells is deposited early during development in BM and could be a source of pluripotent stem cells for tissue/ organ regeneration.

Background:The results from small clinical studies suggest that therapy with adult bone marrow (BM)derived cells (BMCs) reduces infarct size and improves left ventricular function and perfusion. However, the effects of BMC transplantation in patients with ischemic heart disease remains unclear.Methods: We searched MEDLINE, EMBASE, Science Citation Index, CINAHL (Cumulative Index to Nursing and Allied Health), and the Cochrane Central Register of Controlled Trials (CENTRAL) (through July 2006) for randomized controlled trials and cohort studies of BMC transplantation to treat ischemic heart disease. We conducted a random-effects meta-analysis across eligible studies measuring the same outcomes.Results: Eighteen studies (N = 999 patients) were eligible. The adult BMCs included BM mononuclear cells, BM mesenchymal stem cells, and BM-derived circulat-ing progenitor cells. Compared with controls, BMC transplantation improved left ventricular ejection fraction (pooled difference, 3.66%; 95% confidence interval [CI], 1.93% to 5.40%; PϽ.001); reduced infarct scar size (−5.49%; 95% CI, −9.10% to −1.88%; P=.003); and reduced left ventricular end-systolic volume (−4.80 mL; 95% CI, −8.20 to −1.41 mL; P=.006). Conclusions:The available evidence suggests that BMC transplantation is associated with modest improvements in physiologic and anatomic parameters in patients with both acute myocardial infarction and chronic ischemic heart disease, above and beyond conventional therapy. Therapy with BMCs seems safe. These results support conducting large randomized trials to evaluate the impact of BMC therapy vs the standard of care on patient-important outcomes.

Background Despite rapid clinical translation and widespread enthusiasm, the therapeutic benefits of adult bone marrow cell (BMC) transplantation in patients with ischemic heart disease (IHD) continue to remain controversial. A synthesis of the available data is critical to appreciate and underscore the true impact of this promising approach. Methods and Results A total of 50 studies (enrolling 2,625 patients) identified by database searches through January 2012 were included. Weighted Mean Differences for changes in left ventricular (LV) ejection fraction (LVEF), infarct size, LV end-systolic volume (LVESV), and LV end-diastolic volume (LVEDV) were estimated using random effects meta-analysis. Compared with controls, BMC-treated patients exhibited greater LVEF (3.96%, 95% confidence interval (CI): 2.90, 5.02; P<0.00001), and smaller infarct size (–4.03%, CI: –5.47, –2.59; P<0.00001), LVESV (–8.91 ml, CI: –11.57, –6.25; P<0.00001), and LVEDV (–5.23 ml, CI: – 7.60, –2.86; P<0.0001). These benefits were noted irrespective of the study design (RCT vs. Cohort study) and the type of IHD (acute myocardial infarction vs. chronic IHD), and persisted during long-term follow-up. Importantly, the all-cause mortality, cardiac mortality, and the incidence of recurrent MI and stent thrombosis were significantly lower in BMC-treated patients compared with controls. Conclusions Transplantation of adult BMCs improves LV function, infarct size, and remodeling in patients with IHD compared with standard therapy, and these benefits persist during long-term follow-up. BMC transplantation also reduces the incidence of death, recurrent MI, and stent thrombosis in patients with IHD.

Recently, we purified from adult murine bone marrow (BM) a population of CXCR4À very small embryonic-like (VSEL) stem cells and hypothesized that similar cells could be also present in human cord blood (CB). Here, we report that by employing a novel two-step isolation procedure -removal of erythrocytes by hypotonic lysis combined with multiparameter sorting -we could isolate from CB a population of human cells that are similar to murine BM-derived VSELs, described previously by us. These CBisolated VSELs (CB-VSEL) are very small (3-5 lm) and highly enriched in a population of CXCR4À CB mononuclear cells, possess large nuclei containing unorganized euchromatin and express nuclear embryonic transcription factors Oct-4 and Nanog and surface embryonic antigen SSEA-4. Further studies are needed to see if human CB-isolated VSELs similar to their murine BM-derived counterparts are endowed with pluripotent stem cell properties.

Proper response of normal stem cells (NSC) to motomorphogens and chemoattractants plays a pivotal role in organ development and renewal/regeneration of damaged tissues. Similar chemoattractants may also regulate metastasis of cancer stem cells (CSC). Growing experimental evidence indicates that both NSC and CSC express G-protein-coupled seven-transmembrane span receptor CXCR4 and respond to its specific ligand a-chemokine stromal derived factor-1 (SDF-1), which is expressed by stroma cells from different tissues. In addition, a population of very small embryonic-like (VSEL) stem cells that express CXCR4 and respond robustly to an SDF-1 gradient was recently identified in adult tissues. VSELs express several markers of embryonic and primordial germ cells. It is proposed that these cells are deposited early in the development as a dormant pool of embryonic/pluripotent NSC. Expression of both CXCR4 and SDF-1 is upregulated in response to tissue hypoxia and damage signal attracting circulating NSC and CSC. Thus, pharmacological modulation of the SDF-1-CXCR4 axis may lead to the development of new therapeutic strategies to enhance mobilization of CXCR4 þ NSC and their homing to damaged organs as well as inhibition of the metastasis of CXCR4 þ cancer cells.

We reported that complement cascade (CC) becomes activated in bone marrow (BM) during granulocyte colony stimulating factor (G-CSF) mobilization of hematopoietic stem/progenitor cells (HSPCs) and demonstrated that while the third CC component (C3)-deficient mice are easy mobilizers, the fifth CC component (C5)-deficient mice mobilize very poorly. To explain this, we postulated that activation/cleavage of CC releases C3a and C5a anaphylatoxins that differently regulate mobilization. Accordingly, C3a, by enhancing responsiveness of HSPCs to decreasing concentrations of stromal-derived growth factor-1 (SDF-1) in BM, prevents mobilization and promotes their BM-retention. As such, we focused on the mobilization-enhancing role of C5a herein. We found that C5a receptor (C5aR) is not expressed on the surface of HSPCs, and C5a-mediated pro-mobilization effects are mediated by stimulation of granulocytes. Overall, our data support a following model. First C5aR+ granulocytes are chemoattracted by plasma C5 cleavage fragments, being the first wave of cells leaving BM. This facilitates subsequent egress of HSPCs. In the next step, after leaving the BM, granulocytes undergo degranulation in response to plasma C5a and secrete some cationic peptides (cathelicidin, β-defensin) that as demonstrated here for a first time highly enhance responsiveness of HSPCs to plasma SDF-1 gradient. In conclusion, our data reveal the underappreciated central role of innate immunity in mobilization where C5 cleavage fragments via granulocytes orchestrate this process.

Stem cell therapy is being intensely investigated within the last years. Expectations are high regarding mesenchymal stem cell (MSC) treatment in translational medicine. However, many aspects concerning MSC therapy should be profoundly defined. Due to a variety of approaches that are investigated, potential effects of stem cell therapy are not transparent. On the other hand, most results of MSC administration in vivo have confirmed their safety and showed promising beneficial outcomes. However, the therapeutic effects of MSC-based treatment are still not spectacular and there is a potential risk related to MSC applications into specific cell niche that should be considered in long-term observations and follow-up outcomes. In this review, we intend to address some problems and critically discuss the complex nature of MSCs in the context of their effective and safe applications in regenerative medicine in different diseases including graft versus host disease (GvHD) and cardiac, neurological, and orthopedic disorders.