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Summary Hematopoietic stem and progenitor cell (HSPC) mobilization into the blood occurs under normal physiological conditions and is stimulated in the clinic to enable the isolation of HSPCs for transplantation therapies. In the present study, we identify the tetraspanin CD82 as a novel regulator of HSPC mobilization. Using a global CD82 knockout (CD82KO) mouse, we measure enhanced HSPC mobilization after granulocyte-colony stimulating factor (G-CSF) or AMD3100 treatment, which we find is promoted by increased surface expression of the sphingosine 1-phosphate receptor 1 (S1PR1) on CD82KO HSPCs. Additionally, we identify a disruption in S1PR1 internalization in CD82-deficient HSPCs, suggesting that CD82 plays a critical role in S1PR1 surface regulation. Finally, combining AMD3100 and anti-CD82 treatments, we detect enhanced mobilization of mouse HSPCs and human CD34+ cells in animal models. Together, these data provide evidence that CD82 is an important regulator of HSPC mobilization and suggests exploiting the CD82 scaffold as a therapeutic target to enhance stem cell isolation., Graphical abstract, Highlights • CD82 knockout enhances hematopoietic stem and progenitor cell (HSPC) mobilization • CD82 knockout promotes HSPC mobilization through an S1PR1-mediated mechanism • CD82 knockout increases S1PR1 expression by disrupting internalization • Anti-CD82 treatment enhances mobilization of mouse HSPCs and human CD34+ cells, Saito-Reis et al. discovers that loss of the tetraspanin CD82 enhances hematopoietic stem and progenitor cell (HSPC) mobilization into blood through an S1PR1-mediated mechanism. Moreover, they demonstrate that anti-CD82 treatments promote increased mobilization of mouse HSPCs and human CD34+ cells in animal models. Collectively, this work suggests exploiting the CD82 scaffold as a novel therapeutic target to enhance HSPC isolation.

We provide evidence to support the use of TotiCyte as a novel volume reduction technology capable of significantly improving CD34+ stem cell recovery

Over the last decade research on cancer stem cells (CSC) significantly contributed to a better understanding of tumor biology. Given their similarity to normal stem cells, i.e. self-renewal and pluripotency the need arises to develop robust protocols for the isolation and characterization of CSCs. As with other malignancies, hepatic tumors are composed of a heterogeneous population of cells including liver cancer stem cells (LCSC). Yet, a precise understanding of why stem cells become cancerous is still lacking. There is unmet need to develop robust protocols for the successful isolation of LCSCs from human tissue resection material as to assist in the development of molecular targeted therapies. Here we review the research progress made in the isolation and characterization of LCSCs by considering a wide range of cell surface markers and sorting methods, as applied to side populations, microsphere cultures and the gradient centrifugation method. We emphasize the different fluorescence activated cell sorting methods and the possibility to enrich LCSCs by immunomagnetic beads. We review the specificity of functional assays by considering ABCG transporter and ALDH1 enzyme activities and evaluate the in vivo tumorigenicity of LCSCs in highly sensitive bioassays. Finally, we evaluate different LCSC markers in association with viral and non-viral liver disease and explore the potential of novel drug delivery systems targeting CD133, EpCAM, CD13 and CD90 for the development of molecular targeted therapies. Graphical Abstract.

Mesenchymal Stromal/Stem Cells (MSCs) were originally discovered in the 1970s. MSCs are considered a multipotent population that retain tri-lineage differentiation. These cells can be sourced from a variety of tissues, including bone marrow, adipose, molar pulp, and birth tissues. Historically varying definitions of MSCs have existed, but in 2006, the International Society for Cellular Therapy (ISCT) set forth a standardized, minimal criteria for MSCs, including plastic adherence, differentiation into osteoblasts, adipocytes, and chondrocytes, as well as the presence of specific cell surface markers (CD73, CD90, CD105), the absence of hematopoietic surface markers (CD34, CD45, CD11b, to name a few) and a lack or low expression of MHC Class II markers assessed via flow cytometry. In the current chapter, cell isolation and culturing methods from two differing birth tissue sources of MSCs are discussed. Cell isolation is described using tissue excision and migration as well as enzymatic digestion and plating. Histological and cellular identification markers were used along with flow cytometry applications, as outlined by the ISCT. Proliferation potential and growth kinetics are evaluated, and a description of trilineage differentiation has been provided. Lastly, a brief outline of the regenerative potential using MSCs in clinical applications is discussed.

Skeletal muscle is among the most age-sensitive tissues in mammal organisms. Significant changes in its resident stem cells (i.e., satellite cells, SCs), differentiated cells (i.e., myofibers), and extracellular matrix cause a decline in tissue homeostasis, function, and regenerative capacity. Based on the conservation of aging across tissues and taking advantage of the relatively well-characterization of the myofibers and associated SCs, skeletal muscle emerged as an experimental system to study the decline in function and maintenance of old tissues and to explore rejuvenation strategies. In this review, we summarize the approaches for understanding the aging process and for assaying the success of rejuvenation that use skeletal muscle as the experimental system of choice. We further discuss (and exemplify with studies of skeletal muscle) how conflicting results might be due to variations in the techniques of stem cell isolation, differences in the assays of functional rejuvenation, or deciding on the numbers of replicates and experimental cohorts.

BACKGROUND: Adipose tissue-derived stem cells are of great interest because of their properties of immune modulation, tissue regeneration, and multipotent differentiation. To advance development of stem cell-based treatments, determination of the physiologic concentration of adipose tissue-derived stem cells in human adipose tissue is relevant for proper guidance of stem cell treatment dosage, oncologic safety, and evaluation of efficacy.METHODS: A prospective comparative case-control study of 20 patients was conducted to determine the yield of adipose tissue-derived stem cells in periumbilical adipose tissue harvested by the widely used method of aspiration and in structurally intact adipose tissue harvested by excision. Stem cells were isolated using conventional enzymatic digestion and by a method combining enzymatic digestion with mechanical distortion. Stem cell yield was quantified by multicolor flow cytometry and colony-forming capacity.RESULTS: When only the conventional enzymatic digestion was used, no significant difference in adipose tissue-derived stem cell yield was observed. However, when enzymatic digestion was combined with mechanical distortion, twice as many stem cells were isolated from excised adipose tissue compared to aspirated adipose tissue. Inclusion of mechanical distortion significantly increased yield 5-fold in excised adipose tissue and 2-fold in aspirated adipose tissue. Combining enzymatic digestion and mechanical distortion, measured levels of excised adipose tissue reached 140 × 10 (95 percent CI, 62 to 220 × 10) adipose tissue-derived stem cells per gram of adipose tissue that corresponded to 26 × 10 (95 percent CI, 18 to 33 × 10) colony-forming units per gram.CONCLUSIONS: The study indicates that harvesting by aspiration halves the concentration of adipose tissue-derived stem cells in adipose tissue samples when compared to structural intact adipose tissue. Furthermore, the study presents stem cell yield higher than previously described in the current literature.CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Background Bone tissue is one of the tissues that are capable of self-regeneration. However, bone self-regeneration is defeated in the case of broad lesion of bone structure. Isolated stem cells from wisdom tooth follicles can potentially differentiate into ectodermal and mesodermal cells. Saghez is a natural substance that has been extracted from Pistacia terebinthus with unique features, such as high temperature and mechanical stability, adhesive structure, biocompatibility, and anti-neoplastic properties. Methods In this study, Saghez-encapsulated BMP2 was applied as a scaffold for wisdom tooth follicle stem cell differentiation into the osteocyte. A total of three wisdom tooth follicles were obtained for stem cell isolation. For verification of differentiation of the isolated stem cells into osteocyte and adipocyte, Oil Red and Alizarin staining were applied, respectively. Moreover, mesenchymal stem cells were distinguished by profiling their cell surface markers, includingCD73, CD90, CD44, and CD105, by flow cytometry. Saghez scaffold loaded with BMP2 factor was prepared using sol-gel method. Four experimental groups were considered in this study: cells seeded on BMP2 encapsulated in Saghez scaffold, Saghez scaffold, osteogenic medium, and DMEM medium. Results Mechanical properties of Saghez scaffold, including tensile Young’s modulus, ultimate tensile stress, compression Young’s modulus, and complex shear modulus, were 19 MPa, 32 MPa, 0.42 MPa, and 0.9 MPa, respectively. The porosity of the scaffold was 70–140 μm, and the percentage of porosity was 75–98%. The results of flow cytometry studies indicated that CD44, CD73, CD90, and CD105 were positively expressed on the membrane of the tooth follicles’ stem cell. The results indicated that the rate of differentiation of the follicle stem cells into osteocyte was the highest in the Saghez-BMP2 scaffold containing differentiation medium groups. These findings were verified by morphological studies, osteoblast and osteocalcin gene and protein expression investigations, and alkaline phosphatase activity measurement. The highest osteopontin and osteocalcin genes expression levels (1.7 and 1.9) were seen in positive control, followed by DMEM + differentiation factor (1.5 and 1.6), scaffold + BMP2 (1.2 and 1.4), DMEM + stem cell (1 and 1) and scaffold (0.4 and 0.5), and negative control respectively. Conclusion This study provides a novel system for differentiation of the stem cell into osteocytes. The results of this study suggest that loaded BMP2 in Saghez scaffold possibly acts as an osteocyte differentiator factor.

Introduction: The efficiency of stem cell isolation, culture, and biological characterization techniques for treatment is facing serious challenges. The purpose of this study was to provide a protocol for isolation and culture of three types of mesenchymal stem cells (MSCs) derived from the human placenta, amniotic membrane, and umbilical cord with high efficiency used for cell therapy.Methods: During this experimental laboratory study, 10 complete placenta samples were prepared from cesarean section mothers. The protocol for isolation and culture of mesenchymal cells from the placenta tissue, umbilical cord, and amniotic membrane was enzymatically optimized. The morphological features of mesenchymal cells were investigated using an inverted microscope and their biological features were measured using flow cytometry. The differentiation potential of the cells was evaluated by measuring their differentiation capacity into osteocytes and adipocytes. The absorption and reflectance features of the cells were recorded by optical spectroscopy. Finally, the data were statistically analyzed.Results: The expression of CD44, CD73, CD90, and CD29 markers in human placenta tissue-derived cells was significant. CD14, CD34, and CD45 markers were not expressed or were slightly expressed. These cells were highly viable and successfully differentiated into osteocytes and adipocytes. MSCs absorbed more light than visible light by showing light absorption peaks at wavelengths of about 435 and 550 nm.Conclusion: The protocol used in this study for isolation and culture of human placenta tissue-derived MSCs had significant efficiency for the production of MSCs for use in cell therapy and tissue engineering. DOI: 10.34172/jlms.2021.18

Summary Trophoblast cells are the first differentiated cells formed from a fertilized egg during mammalian development, and they secrete several autocrine and paracrine factors essential for sustaining pregnancy. In pathological conditions, these cells secrete various proinflammatory cytokines affecting both maternal and fetal health. Here, we provide a detailed protocol for isolation, maintenance, differentiation, and detection of factors secreted from trophoblast stem (TS) cells. This protocol provides conditions for inducing genotoxic stress in differentiated TS cells and detecting the effects on cytokine production. For complete details on the use and execution of this protocol, please refer to Singh et al. (2020)., Graphical Abstract, Highlights • Describes mouse trophoblast stem cell isolation and differentiation protocol • Provides details for inducing genotoxic stress in differentiated TS cells • Provides methodology to detect factors secreted from differentiated TS cells, Trophoblast cells are the first differentiated cells formed from a fertilized egg during mammalian development, and they secrete several autocrine and paracrine factors essential for sustaining pregnancy. In pathological conditions, these cells secrete various proinflammatory cytokines affecting both maternal and fetal health. Here, we provide a detailed protocol for isolation, maintenance, differentiation, and detection of factors secreted from trophoblast stem (TS) cells. This protocol provides conditions for inducing genotoxic stress in differentiated TS cells and detecting the effects on cytokine production.

Genomic instability, incurred during the process of stem cell isolation, culture expansion, and reprogramming remains the most critical determinant that has hampered the establishment of a stem cell-based therapeutic approach as a viable option in the clinical perspective. The reported divergence in the efficiency of survival, proliferation, differentiation, and pluripotency may be attributed to genetic damage experienced by the donor cells during their in vitro handling. The genotoxic stress continues or even gets exacerbated during in vitro manipulations before their theranostic applications. Moreover, the genomic instability observed in the cells is cell type-specific, being different between induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) from bone marrow or adipose origin among others. Moreover, the biological characteristics of the in vitro manipulated stem cells are determined by the extent of DNA damage, variation in the mechanism of homologous and non-homologous end-joining repair, telomere shortening, and senescence resulting from replication stress, chromosomal rearrangements, and point mutations. Additionally, epigenetic instability needs to be concisely considered. The donor and recipient characteristics like age, medical or environmental exposure, and co-morbidities require special consideration. Our chapter in-depth reviews the contributing factors that influence the genomic stability of stem cells thus impeding the therapeutic goals in general, and favor the development of adverse effects, i.e., risk of tumorigenicity. The relevance of the specific aneugenic changes or structural chromosome aberrations aroused in the culture and the time-lapse until their occurrence and implications for gene dysregulation will be discussed. Finally, the methods in use or those being proposed for to their applicability in DNA damage evaluation will be explained with a primary focus in their sensitivity to scrutinize DNA changes.

Stem cell therapy promotes tissue regeneration and wound healing. Efforts have been made to prime stem cells to enhance their regenerative abilities. Certain marijuana components, namely the non-psychoactive cannabidiol (CBD) and psychoactive tetrahydrocannabinol (THC), are defined as immunomodulators.9 We test whether two sources of stem cells, primed with CBD or THC, would demonstrate improved regenerative abilities. Human adipose-derived stem cells (ASCs) and bone marrow-derived stem cells (BMDSCs), not obtained from the same individual, were treated with low (300 nM) or high (3 μM) concentration CBD. Porcine ASCs and BMDSCs were isolated from a single pig, and treated with either low or high concentrations of CBD or THC. Transwell migration and MTT proliferation assays were performed on the human ASCs and BMDSCs. Also, transwell migration assay was performed on the porcine ASCs and BMDSCs. Finally, a wound healing scratch assay in porcine primary fibroblasts (PFs) was performed, co-cultured with the cannabinoid-treated ASCs. CBD priming at low concentration induces migration by 180% (P < .01) in porcine ASCs, and by only 93% (P < .02) in porcine BMDSCs. In porcine stem cells, THC priming at low concentration induces migration by 91.6% (P < .01) in ASCs but by only 44.3% (P < .03) in BMDSCs. Compared to PFs co-cultured with untreated ASCs, PFs co-cultured with low CBD-primed ASCs had 75% faster wound closure at 18 hours (P < .01). CBD and THC priming of ASCs and BMDSCs, particularly at lower doses, enhances a number of regenerative parameters, suggesting that these major marijuana components may improve stem cell-based therapies. SIGNIFICANCE OF THE STUDY: Our study demonstrates that cannabinoids can enhance the regenerative capacity of two major sources of stem cells, adipose- and bone marrow-derived, from human and porcine donors. Stem cell isolation and expansion is invasive, costly and time consuming. Stem cells with improved regenerative properties may be effective in the treatment of acute or chronic wounds. This is the first study to compare the priming potential of two sources of stem cells from the same animal, with the same genetic and epigenetic profile, as well as the first to prime with THC.

Background Lipofilling is one of the most often performed surgical procedures in plastic and reconstructive surgery. Lipoaspirates provide a ready source of stem cells and secreted factors that contribute to neoangiogenesis and fat graft survival. However, the regulations about the enrichment of these beneficial cells and factors are ambiguous. In this study, the authors tested whether a combination of centrifugation and homogenization allowed the enrichment of viable stem cells in lipoaspirates through the selective removal of tumescent solution, blood, and released lipids without significantly affecting the cell secretome. Methods Human lipoaspirate was harvested from six different patients using water jet-assisted liposuction. Lipoaspirate was homogenized by first centrifugation (3584 rpm for 2 minutes), shear strain (10 times intersyringe processing), and second centrifugation (3584 rpm for 2 minutes). Stem cell enrichment was shown by cell counting after stem cell isolation. Lipoaspirate from different processing steps (unprocessed, after first centrifugation, after homogenization, after second centrifugation) was incubated in serum-free cell culture medium for mass spectrometric analysis of secreted proteins. Results Lipoaspirate homogenization leads to a significant 2.6 ± 1.75-fold enrichment attributable to volume reduction without reducing the viability of the stem cells. Protein composition of the secretome did not change significantly after tissue homogenization. Considering the enrichment effects, there were no significant differences in the protein concentration of the 83 proteins found in all processing steps. Conclusions Stem cells can be enriched mechanically without significantly affecting the composition of secreted proteins. Shear-assisted enrichment of lipoaspirate constitutes no substantial manipulation of the cells' secretome.

Production technology and characteristics of the cell product obtained using the ‘ESVIEF System’ kit (developed by JoinTechCell LLC, Russian Federation) for isolation of a stromal vascular fraction from human adipose tissue are described. The use of subcutaneous fat as a source of stem and progenitor cells for regenerative medicine has become widespread during the last decade. The main advantage of using adipose tissue as a source of stem cells when compared with bone marrow is the lower invasiveness of the material sampling procedure during liposuction in addition to the significantly larger number of multipotent mesenchymal stromal cells obtained per unit of tissue volume. The development and implementation of devices for automation and standardisation of stem cell isolation from adipose tissue are important for the widespread use of stem cells in clinical practice. This work aimed to evaluate the effectiveness and safety of the cell production technology using the ‘ESVIEF System’ kit for isolation of a stromal vascular fraction from human adipose tissue. Adipose tissue samples obtained from patients during liposuction were used as clinical material. The obtained cell fractions were studied using microscopy, flow cytometry and cell culture methods. The viability of the stromal vascular fraction cells (nucleated) was 90.9 ± 0.3% with a total number of 0.81 ± 0.08 × 106 cells/ml of adipose tissue. The study showed that the ‘ESVIEF System’ kit that was developed for isolating stromal vascular fractions from human adipose tissue is a promising and safe technology for producing cell products.

Background: Adipose tissue (AT) is a rich source of mesenchymal stem cells (MSCs), however, there is no standardized protocol for stem cell isolation and culture. This leads to inconsistency of the results and limits the comparison of the data from different laboratories. Our aim was to provide an applied protocol for ASCS isolation and expansion, study the cell behavior and define their cellular surface markers. ASCs were cultured from both resected adipose tissue (RAT) obtained following abdominoplasty or breast reduction and lipoaspirates (LPA) following laser-free liposuction. Method: the protocol entailed coculturing of stromal vascular fraction (SVF) with RAT as raw pieces using DMEM medium with varying glucose concentration. The coculture protocol aimed to mimic the normal physiological conditions required for cell growth. ASCs were immunophenotyped to define their MSCs surface markers by flowcytometry. Results: ASCs were isolated from coculturing RAT with SVF with fibroblast-like adherent cells morphology. The ASCs yield isolated from LPA was significantly greater than from RAT on day 14 and 28 (p = 0.002

BACKGROUND: The term amniotic fluid (AF) can be an ideal alternative as a source of mesenchymal stem cells (MSCs), originating from the neonate. Preclinical studies of the second- and third-trimester amnion fluid cells confirmed the number of potential donors from this wasted material. AIM: This study aims to look at the forming time of stem cells derived from AF-MSCs. MATERIALS AND METHODS: AF samples from healthy human donors were collected during full-term C-sections and kept at 4°C until processed. The number of colony-forming unit-fibroblast was assessed microscopically by calculating spindle-shaped colonies that clearly resembled of fibroblasts and did not include colonies with rounded epithelioid morphology. The immunophenotyping of their independent AF preparations was done using the human MSC phenotyping kit which was done according to the manufacturer’s instruction by flow cytometry. RESULTS: The result showed that it succeeded in getting 8 million cells which will be used for research on pelvic organ prolapse therapy using AF-MSCs. The stem cell isolation totally takes 6 weeks. We got 2 million stem cells in one flask. CONCLUSION: This study concludes that the time needed for differentiation of AF-MSCs is 6 weeks and AF-MSCs express mesenchymal markers such as CD90, CD73 (SH3, SH), and CD105 (SH2) and these cells also express HLA antigens – ABC, CD 34, and CD 45 which are hematopoietic markers and endothelial CD31 markers.

Regenerative medicine as a background of stem cell research and therapy has a long history. A wide variety of diseases including Parkinson's disease, heart diseases, multiple sclerosis, spinal cord injury, diabetes mellitus and etc. are candidate to be treated using different types of stem cells. There are several sources of stem cells such as bone marrow, umbilical cord, peripheral blood, germ cells and the embryo/fetus tissues. Fetal stem cells (FSCs) and embryonic stem cells (ESCs) have been described as the most potent stem cell source. Although their pluri- or multipotent properties leads to promising reports for their clinical applications, owning to some ethical and legal obstacles in different communities such as Muslim countries, care should be taken for therapeutic applications of FSCs and ESCs. Derivation of these cell types needs termination of pregnancy and embryo or fetus life that is prohibited according to almost all rules and teaches in Muslim communities. Abortion and termination of pregnancy under a normal condition for the procurement of stem cell materials is forbidden by nearly all the major world religions such as Islam. Legislated laws in the most of Muslim countries permit termination of pregnancy and abortion only when the life of the mother is severely threatened or when continuing pregnancy may lead to the birth of a mentally retarded, genetically or anatomically malformed child. Based on the rules and conditions in Islamic countries, finding an alternative and biologically normal source for embryonic or fetal stem cell isolation will be too difficult. On the one hand, Muslim scientists have the feasibility for finding of genetically and anatomically normal embryonic or fetal stem cell sources for research or therapy, but on the other hand they should adhere to the law and related regional and local rules in all parts of their investigation. The authors suggest that the utilization of ectopic pregnancy (EP) conceptus, extra-embryonic tissues, and therapeutic abortion materials as a valuable source of stem cells for research and medical purposes can overcome limitations associated with finding the appropriate stem cell source. Pregnancy termination because of the mentioned subjects is accepted by almost all Islamic laws because of maternal lifesaving. Also, there are no ethical or legal obstacles in the use of extra-embryonic or EP derived tissues which lead to candidate FSCs as a valuable source for stem cell researches and therapeutic applications.

Cryopreservation of testicular tissue before cancer therapy for fertility preservation in prepubertal boys with cancer is of great interest in reproductive medicine. Isolation of spermatogonial stem cells (SSCs) from cryopreserved tissues would be a suitable cell source to re-establish spermatogenesis after cancer therapy. We herein establish optimized protocols for cryopreservation of human testicular tissue and isolation of SSCs from cryopreserved tissue. We developed a freezing protocol that provided high testicular cell viability and supported structural integrity and tubular epithelium coherence similar to fresh tissue. Then, we established a protocol that allowed efficient isolation of functional SSCs from cryopreserved tissues. Isolated cells were found on the testicular basement membrane after xenotransplantation. Our results demonstrated the preservation of testicular tissue structure and high cell viability with efficient isolation of SSCs after testicular cryopreservation, which is promising for future therapeutic applications in fertility preservation.

Isolation of pure populations of mesenchymal stem cells from bone marrow aspirate is a critical need in regenerative medicine such as orthopedic and cartilage reconstruction with important clinical and therapeutic implications. Currently available stem cell isolation systems mainly rely on intrusive immuno-labeling techniques. Mesenchymal stem cells in bone marrow samples are typically larger than other cells, which can be used as a distinctive biophysical cue for non-invasive isolation. In this work, a spiral-shaped inertial microfluidic sorter was developed to isolate mesenchymal stem cells from mouse bone marrow samples with minimal sample preparation steps. To characterize the sorting performance, cultured mesenchymal stem cells were spiked into tissue-digested mouse bone marrow cells. At a flow rate of 1.6 mL min−1, an average enrichment of 6.0× and a recovery of 73% were demonstrated. About 3 × 106 tissue-digested bone marrow cells can be processed in 1 minute with a single microfluidic run. The recovered mesenchymal stem cells after microfluidic sorting retained high (>95%) viability and similar immuno-phenotype expressions and multi-potencies in tri-differentiation lineages to unprocessed cells. This rapid, label-free and non-invasive inertial microfluidic sorter has practical applications in target stem cell enrichment in stem cell therapy.

Successful isolation of human endometrial stem cells from menstrual blood, namely menstrual blood‐derived endometrial stem cells (MenSCs), has provided enticing alternative seed cells for stem cell‐based therapy. MenSCs are enriched in the self‐regenerative tissue, endometrium, which shed along the periodic menstrual blood and thus their acquisition involves no physical invasiveness. However, the impact of the storage duration of menstrual blood prior to stem cell isolation, the age of the donor, the number of passages on the self‐renewing of MenSCs, the paracrine production of biological factors in MenSCs and expression of adhesion molecules on MenSCs remain elusive. In this study, we confirmed that MenSCs reside in shedding endometrium, and documented that up to 3 days of storage at 4°C has little impact on MenSCs, while the age of the donor and the number of passages are negatively associated with proliferation capacity of MenSCs. Moreover, we found that MenSCs were actually immune‐privileged and projected no risk of tumour formation. Also, we documented a lung‐ and liver‐dominated, spleen‐ and kidney‐involved organic distribution profile of MenSC 3 days after intravenous transfer into mice. At last, we suggested that MenSCs may have potentially therapeutic effects on diseases through paracrine effect and immunomodulation.

Background Adipose-derived stem cells are recognized as being an effective mesenchymal stem cell population with enormous potential in different fields of regenerative medicine and stem cell therapy. Although there is unanimous agreement on the harvesting procedure for adipose tissue, there are various protocols for adipose-derived stem cell isolation. The aim of this study was compare two methods of adipose-derived stem cells (ASCs) isolation, one based on a mechanical + enzymatic (ME) procedure and the other one exclusively mechanical (MC), in order to determine which one was superior to the other in accordance with current European and US legislation. Methods We reported step by step the two different methods ASCs isolation by comparing them. The ME procedure included the use of a centrifuge, an incubator and collagenase digestion solution (Collagenase NB 6 GMP Grade 17458; Serva GmbH, Heidelberg, Germany). The MC procedure was performed by vibrating shaker and centrifuge, both placed in a laminar airflow bench. Results With the ME procedure, a mean of 9.06 × 105 ASCs (range, 8.4 to 9.72 × 105; SD ± 6.6 × 105) was collected, corresponding to 25.9% of the total number of harvested cells. With the MC procedure, a mean of 5 × 105 ASCs (range: 4.0 to 6.0 × 105; SD, ±1 × 105) was collected, corresponding to 5% of the total number of harvested cells. Conclusion Based on data collected, from the same amount of lipoaspirate the ME procedure allowed to isolate a greater number of ASCs (25,9%) compared to the MC one (5%)., Highlights • Adipose-derived stem cells (ASCs) are effective mesenchymal stem cell population with enormous potential. • In this study we compared two methods of adipose-derived stem cells (ASCs) isolation. • Gathered data showed a greater amount of isolated ASCs by the ME procedure as compared to the MC one.

Cardiac regenerative therapy includes several techniques to repair and replace damaged tissues and organs using cells, biomaterials, molecules, or a combination of these factors. Generation of heart muscle is the most important challenge in this field, although it is well known that new advances in stem cell isolation and culture techniques in bioreactors and synthesis of bioactive materials contribute to the creation of cardiac tissue regeneration in vitro. Some investigations in stem cell biology shows that stem cells are an important source for regeneration of heart muscle cells and blood vessels and can thus clinically contribute to the regeneration of damaged heart tissue. The aim of this review was to explain the principles and challenges of myocardial tissue regeneration with an emphasis on stem cells and scaffolds. J. Cell. Biochem. 118: 2454-2462, 2017. © 2017 Wiley Periodicals, Inc.

Stem cell research has great potential in clinical applications and regenerative medicine, which highlight the need for efficient stem cell isolation technologies. Enrichment of stem cells is challenging because of its sensitivity to the surrounding environment and its overlapping features with non-target cells. Current gold-standard isolation techniques depend on surface antigens that are exclusively expressed on stem cells, where surface immuno-labeling is followed by flow cytometry or magnetic sorting procedures. It is well-established that stem cells should be enriched with minimal alteration, therefore, isolation without immunological tagging in a label-free format is of great interest. Microfluidic technologies have demonstrated high level of control to precisely manipulate stem cells. Here, we review the recent emerging label-free microfluidic based cell isolation technologies that provide unbiased stem cell enrichment based on their size and deformability, adhesion, and electrical characteristics, and discuss their potential to achieve effective stem cell isolation for research and clinical applications.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution. HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

This study was aimed to find a practical technique for isolation and culture spermatogonial stem cells from male brown trout (Salmo trutta macrostigma). Twelve wild juvenile male were obtained from Kılıç Trout Fish Farm (Kahramanmaraş, Turkey). The juveniles were taken alive to the aquaria unit and stored in a 1000-liter capacity fiberglass tank. In order to identify the best size, age and testis structure of S.t. macrostigma for spermatogonial stem cell isolation and culture. Morphological and histological testis conditions were assessed. Fish were anesthetized with 0.04% 2-phenoxethanol. The surface of the fish was sterilized with 70% ethanol. Twelve fish were divided into two groups for enzyme digestion, and each group was divided into two replicates (three fish per replicate). Testis tissue of group one were digested by 0.25% trypsin- EDTA, and testis tissues of group two were digested by 0.05% trypsin-EDTA. At the end of the trial, first, the best age, size and weight of the male fish for spermatogonial stem cell isolation and culture were identified as 5+ month old, 12.13±1.5 cm, 19, 25±7.05 g respectively. Then, the highest spermatogonial stem cells were measured in the stage one and two of the testes. Finally, isolation and culture conditions were optimized for male S.t. macrostigma. Spermatogonial stem cell isolation and culture techniques were defined for fish in order to be used in surrogate reproduction technologies and gene transfer systems.

Stem cells from human exfoliated deciduous teeth (SHED) and human dental pulp stem cells (hDPSCs) have emerged as attractive cell sources for bone regeneration. However, the specific teeth and the conditions most suitable for stem cell isolation remain unclear. Therefore, the success rate of SHED and hDPSCs isolation, the patient age and remaining root length in deciduous teeth were evaluated. Successful isolation was defined as when the cell culture was maintained up to the third passage without any contamination or other issues. Remaining tooth length was calculated using the root-to-crown ratio from patient X-rays and compared to the norm value from the literature. The overall successful isolation rate of SHED and hDPSCs was 82% and 70%. The average patient ages at extraction of the deciduous teeth and permanent teeth were 11 years and 9 months, and 22 years and 10 months respectively. In the successful SHED group, the average remaining root length of the anterior deciduous teeth was 71.4%, and that of the deciduous molars was 61.4%. Successful isolation appears to be associated with patient age, length of the remaining root, and also mechanical stress and other factors. Tooth selection criteria need to be identified to improve the success rate.

Objective To evaluate the feasibility of stem cell isolation from falciform fat harvested via laparoscopic morcellation. Study design Pilot study. Animals Eleven client-owned dogs. Methods Falciform was harvested traditionally via laparotomy and laparoscopically via tissue morcellation. Harvested tissue was processed with a commercially available adipose tissue dissociation kit to obtain a stromal vascular fraction (SVF). Cells were subsequently labeled for CD90, CD45, and CD44 cell surface antigens by using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting flow cytometry. CD90+ cells were quantitated, and their viability was assessed with a hemocytometer and a trypan blue exclusion test of cell viability. Results No perioperative complications occurred in dogs undergoing laparoscopic morcellation. Laparoscopically and traditionally harvested samples yielded an average of 0.39 (±0.1) × 106 and 0.33 (±0.1) × 106 CD90+ cells, respectively, per 10 million SVF cells. CD90+ cell viability after MACS was 89% (±11%) for morcellated and 86% (±7%) for traditionally harvested samples. Neither CD90+ cell quantity nor viability was different between samples obtained via traditional laparotomy vs laparoscopic morcellation (P = .38 and P = .63, respectively). Populations of CD90+ cells isolated with each harvest technique had similar CD44 and CD45 expression profiles. Conclusion Viable populations of CD90+ cells with similar CD44/CD45 expression profiles were isolated from laparoscopically morcellated and traditionally harvested falciform tissue. No appreciable morbidity was associated with laparoscopic falciform morcellation. Clinical significance Laparoscopic morcellation is a safe and effective minimally invasive approach to falciform tissue harvest for adipose-derived mesenchymal stem cell isolation.