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Dental pulp is a tissue obtained from pulp chamber of deciduous and permanent tooth which contain stem cells. Stem cell isolation procedure is performed to obtain cells from tissue using enzymatic digestion. The aim of this study is to isolate and observe the morphology of stem cells during passage 0 and passage 3. Dental pulp from deciduous and permanent tooth was enzymatically digested using collagenase Type I and cells obtained were cultured in DMEM-KO that contains 10% fetal bovine serum, 1% antibiotic-antimycotic solution and 0.001× GlutaMax®. During culture, cell morphology was observed under the microscope on day 3, 16 and 33 and captured using cellB software. Giemsa staining was conducted on cells at passage 3. Cells attached at the bottom of the flask on day 3 and started forming small colonies. Cells became confluent after approximately 4 weeks. Both Stem Cells from Deciduous Tooth (SHED) and Human Dental Pulp Stem Cells (hDPSC) exhibited fibroblast-like morphology during passage 0 and passage 3...

Improvements of (retroviral) gene transfer vectors, stem cell isolation and culture techniques as well as transduction protocols eventually resulted not only in the successful genetic modification of cells capable of reconstituting the haematopoietic system in various animal models, but also human beings. This was a conditio sine qua non for the successful application of gene therapy for inherited diseases as meanwhile achieved for severe combined immune deficiencies (SCID-X1, ADA-SCID) and chronic granulomatous disease (CGD). Unexpectedly, in long-term animal experiments as well as in the follow up of patients from the CGD trial, haematopoietic clones bearing insertions in certain gene loci became dominant, which was most apparent in the myeloid blood compartment. Accumulating data strongly suggest that this clonal dominance was due to some growth and/or survival advantage conferred by gene-activating or -suppressing effects of the integrated retroviral vector (insertional mutagenesis). Importantly, such induced clonal dominance seems not to lead to malignant transformation of affected cell clones inadvertently. The latter finding has become the basis for the concept of 'induced haematopoietic stem cells', a potentially powerful tool to investigate genes involved in the regulation of mechanisms underlying competitive advantages of stem cells, but also in the multi-step nature of malignant transformation. Here we discuss promises and open issues of this concept as well as the important question of common insertion sites statistics and its pitfalls.

Recent observations indicate that, in several types of human cancer, only a phenotypic subset of cancer cells within each tumor is capable of initiating tumor growth. This functional subset of cancer cells is operationally defined as the “cancer stem cell” (CSC) subset. Here we developed a CSC model for the study of human colorectal cancer (CRC). Solid CRC tissues, either primary tissues collected from surgical specimens or xenografts established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, were disaggregated into single-cell suspensions and analyzed by flow cytometry. Surface markers that displayed intratumor heterogeneous expression among epithelial cancer cells were selected for cell sorting and tumorigenicity experiments. Individual phenotypic cancer cell subsets were purified, and their tumor-initiating properties were investigated by injection in NOD/SCID mice. Our observations indicate that, in six of six human CRC tested, the ability to engraft in vivo in immunodeficient mice was restricted to a minority subpopulation of epithelial cell adhesion molecule (EpCAM) high /CD44 + epithelial cells. Tumors originated from EpCAM high /CD44 + cells maintained a differentiated phenotype and reproduced the full morphologic and phenotypic heterogeneity of their parental lesions. Analysis of the surface molecule repertoire of EpCAM high /CD44 + cells led to the identification of CD166 as an additional differentially expressed marker, useful for CSC isolation in three of three CRC tested. These results validate the stem cell working model in human CRC and provide a highly robust surface marker profile for CRC stem cell isolation.

Stem cell biology is one of the most exciting subjects in life science nowadays. The major point in stem cell biology is the extraordinary capacity of these cells to self-renew and to give rise to different cell types. Nevertheless, major issues remain to be cleared and very few diseases can actually be cured based on stem cell therapy. Adult stem cells remain difficult to locate, isolate and amplify in a homogeneous fashion and, thus, limit their therapeutic application in clinical trial. Embryonic stem cells could represent a new hope in stem cell therapy but in addition to the scientific difficulties, over ethical and judiciary issues should be addressed. In order to cure routinely patients, controlled conditions for stem cell isolation, amplification, differentiation, and administration must be defined and effective tissue integration have to be established. In this review we will discuss these different aspects of stem cell biology.

Circumcision is described as a cultural, medical, and religious process which states surgical removal of the foreskin either partly or fully. Cells isolated from the circumcised tissues are referred as foreskin cells. They have been thought as feeder cell lines for embryonic stem cells. Their fibroblastic properties were also utilized for several experiments. The waste tissues that remain after the circumcision thought to have stem cell properties. Therefore, there have been very few attempts to expose their stem cell properties without turning them into induced pluripotent stem cells. Although stem cell isolation from prepuce and their mesenchymal multilineage differentiation potential have been presented many times in the literature, the current study explored hematopoietical phenotype of newborn foreskin stem cells for the first time. According to the results, human newborn foreskin stem cells (hnFSSCs) were identified by their capability to turn into all three germ layer cell types under in vitro conditions. In addition, these cells have exhibited a stable phenotype and have remained as a monolayer in vitro. hnFSSCs suggested to carry different treatment potentials for bone damages, cartilage problems, nerve damages, lesion formations, and other diseases that are derive from mesodermal, endodermal, and ectodermal origins. Owing to the location of the tissue in the body and differentiation capabilities of hnFSSCs, these cells can be considered as easily obtainable and utilizable even better than the other stem cell sources. In addition, hnFSSCs offers a great potential for tissue engineering approaches due to exhibiting embryonic stem cell-like characteristics, not having any ethical issues, and teratoma induction as in embryonic stem cell applications. © 2015, Springer Science+Business Media New York.

Stem cell research has led to many remarkable achievements in recent years, but progress in the study of spermatogonial stem cells (SSCs) has been relatively slow, partly due to the slow development of techniques for spermatogonial stem cell isolation. The major accomplishments of SSC sorting and identification occurred approximately 10 years ago, and since that time, these techniques have been widely used without major improvements. In this article, we briefly introduce the biological properties of SSCs before reviewing the development of sorting techniques for SSCs in the past decades. We then summarize recent achievements in SSC sorting and finally discuss the advantages and disadvantages of SSC isolation methods, to provide new insight into techniques and research related to spermatogonial stem cells and promote the development of reproductive biology.

PURPOSE. Because of a shortage of fresh corneal tissue for research, it was of interest to investigate the potential of successfully isolating human limbal epithelial cells (hLECs) from organ culture corneal-scleral (OCCS) rims. METHODS. Superficial segments of corneal limbus were dissected and digested using collagenase (0.5 mg/mL, 16 hours at 378C). Cell suspensions were separated into four different growth conditions: corneal epithelial cell medium (CM); CM þ 3T3-Swiss albino cells; stromal stem cell medium (SM); and SM þ 3T3 cells. Colony number, hLEC count, cell density, and colony forming efficiency (CFE) were quantified to assess different growth conditions. The expression profile associated with basal hLECs was assessed by immunofluorescence, and epithelial integrity was measured using our real architecture for 3D tissue (RAFT) corneal tissue equivalent. RESULTS. Human limbal epithelial cells can be successfully isolated from OCCS rims following 4 weeks in storage with an 80.55% success rate with 36 corneal rims. Stromal stem cell medium þ 3T3s provided optimal growth conditions. Colony number, total cell number, and cell density were significantly higher at day 7 in cultures with SM than in CM. There were no significant differences between SM and CM when assessing CFE and the expression profile associated with basal hLECs. Cells maintained in SM were found to produce a higher quality epithelium than that cultured in CM. CONCLUSIONS. Organ culture corneal-scleral rims can be a valuable source for hLEC. Using a combination of collagenase-based isolation and medium designed for stromal stem cell isolation, a high number of good quality hLECs can be cultured from tissue that would have otherwise been ignored.

Hematopoietic stem cells (HSCs) have enormous potential for use in transplantation and gene therapy. However, the frequency of repopulating HSCs is often very low; thus, highly effective techniques for cell enrichment and maintenance are required to obtain sufficient cell numbers for therapeutic use and for studies of HSC physiology. Common methods of HSC enrichment use antibodies recognizing HSC surface marker antigens. Because antibodies are known to alter the physiology of other cell types, we investigated the effect of such enrichment strategies on the physiology and lineage commitment of HSCs. We sorted HSCs using a method that does not require antibodies: exclusion of Hoechst 33342 to isolate side population (SP) cells. To elucidate the effect of antibody binding on this HSC population, we compared untreated SP cells with SP cells treated with the Sca-1+c-Kit+Lin− (SKL) antibody cocktail prior to SP sorting. Our findings revealed that HSCs incubated with the antibody cocktail had decreased expression of the stem cell-associated genes c-Kit, Cd34, Tal-1, and Slamf1 relative to untreated SP cells or to cells treated with polyclonal isotype control antibodies. Moreover, SKL antibodies induced cycling in SP cells and diminished their ability to confer long-term hematopoietic engraftment in lethally irradiated mice. Taken together, these data suggest that antibody-based stem cell isolation procedures can have negative effects on HSC physiology.

When male germ line stem cells are transplanted from the testis of a fertile donor animal to the testis of an infertile recipient they can establish donor-derived spermatogenesis in the recipient testis, and the resulting sperm can transmit the genotype of the donor to the offspring of the recipient. Germ cell transplantation provides a bioassay to study the biology of these stem cells, to develop systems for spermatogonial stem cell isolation and culture, to examine defects in spermatogenesis and to correct male infertility. Although most widely studied in rodents, germ cell transplantation has been applied to larger mammals, including primates. A potential clinical application is restoration of fertility in patients that underwent cytotoxic treatments for cancer. As an alternative to transplantation of isolated germ cells to a recipient testis, ectopic grafting of testis tissue from diverse mammalian donor species, including primates, into a mouse host represents a novel possibility to study spermatogenesis, to investigate the effects of toxins or drugs with the potential to enhance or suppress male fertility, and to produce fertile sperm from immature donors. Therefore, transplantation of germ cells or xenografting of testis tissue are uniquely valuable approaches for the study, preservation and manipulation of male fertility in mammalian species.

Diverse in vivo studies have suggested that adult stem cells might have the ability to differentiate into cell types other than those of the tissues in which they reside or derive during embryonic development. This idea of stem cell "plasticity" has led investigators to hypothesize that, similar to embryonic stem cells, adult stem cells might have unlimited tissue regenerative potential in vivo, and therefore, broad and novel therapeutic applications. Since the beginning of these observations, our group has critically examined these exciting possibilities for mouse bone marrow-derived cells by taking advantage of well-characterized models of tissue regeneration, Cre/lox technology, and novel stem cell isolation protocols. Our experimental evidence does not support plasticity of hematopoietic stem cells as a frequent physiological event, but rather indicates that cell fusion could account for reported cases of hematopoietic stem cell plasticity or "transdifferentiation" in vivo. Our studies highlight the need for meticulous technical controls during the isolation, transplantation, tracking, and analysis of bone marrow-derived cells during in vivo studies on plasticity. Further studies will be necessary to better define experimental conditions and criteria to unequivocally prove or reject plasticity in vivo. In this review, we focus on results from several studies from our laboratory, and discuss their conclusions and implications.

Technologies to isolate colonies of human pluripotent stem cells from other cell types in a high-throughput manner are lacking. A microfluidic-based approach that exploits differences in the adhesion strength between these cells and a substrate may soon fill the gap.

The isolation of murine embryonic stem (ES) cells has been almost exclusively from the 129 mouse strain. Other mouse strains, such as CBA, have proven refractory to ES cell isolation by conventional means. We previously reported the isolation of 87.5% CBA ES lines by selective ablation of differentiating cells (McWhir et al., 1996). Here, we report the isolation of ES and EG cells from 94% CBA embryos hemizygous and homozygous for a neomycin-resistance transgene under the transcriptional control of the Oct3/4 promoter (Oct/neo). Since expression of the Oct/neo transgene only confers drug resistance to undifferentiated cells of the inner cell mass, selection results in the ablation of differentiating cells from the culture. The efficiency of ES isolation by selective ablation in homozygotes is twice that in heterozygotes. ES isolation frequency in permissive strain 129 embryos is enhanced by treatment with an inhibitor of the extracellular-signal-regulated kinase (ERK) pathway but this effect is not sufficient to permit ES isolation from the CBA strain.

We examined cell isolation, viability, and growth in adipose-derived stem cells harvested from whole adipose tissue subject to different cryopreservation lengths (2–1159 days) from patients of varying ages (26–62 years). Subcutaneous abdominal adipose tissue was excised during abdominoplasties and was cryopreserved. The viability and number of adipose-derived stem cells isolated were measured after initial isolation and after 9, 18, and 28 days of growth. Data were analyzed with respect to cryopreservation duration and patient age. Significantly more viable cells were initially isolated from tissue cryopreserved 2 years, irrespective of patient age. However, this difference did not persist with continued growth and there were no significant differences in cell viability or growth at subsequent time points with respect to cryopreservation duration or patient age. Mesenchymal stem cell markers were maintained in all cohorts tested throughout the duration of the study. Consequently, longer cryopreservation negatively impacts initial live adipose-derived stem cell isolation; however, this effect is neutralized with continued cell growth. Patient age does not significantly impact stem cell isolation, viability, or growth. Cryopreservation of adipose tissue is an effective long-term banking method for isolation of adipose-derived stem cells in patients of varying ages.

Objective. Although several types of stem cells have been isolated from rodent and human tissues, very few data exist on stem cell isolation from nonrodent animals, which seriously limits the advancement of stem cell biology and its ultimate translation to human clinical applications. Domestic cats are used frequently in biomedical research and are the preferred species for studies of normal physiology and disease, particularly in neuroscience. Therefore, the objective of this study was to characterize mesenchymal stem cells (MSC) from feline bone marrow for use in research on the application of stem cells to human health problems for which cats are the preferred model. Methods. Mesenchymal stem cells from feline bone marrow were isolated by standard methodology developed for other species and characterized according to morphology, growth traits, cell-surface antigen profile, and differentiation repertoire in vitro. Results. Feline mesenchymal stem cells exhibit a fibroblast-like morphology with bipolar or polygonal cell bodies and possess a cell-surface antigen profile similar to their rodent and human counterparts. Feline MSC exist at a frequency of 1 in 3.8 � 10 5 bone marrow mononuclear cells and are capable of differentiation to adipocytic, osteocytic, and neuronal phenotypes when exposed to appropriate induction media. Conclusions. Mesenchymal stem cells isolated from feline bone marrow possess several traits typical of MSC from other species. Characterization of feline mesenchymal stem cells will facilitate future studies of stem cell biology and therapeutics for which the domestic cat is an indispensable model. © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Isolation and purification of stem cells and their delivery into diseased or aged tissues or organs need special devices for proper transplantation of stem cells in order to achieve high cell retention at transplant site for repair or regeneration of tissues and organs. The clinical and preclinical importance of special devices such as Celution System, Isolex cell separation device, magnetic surface-enhanced Raman spectroscopic dots, microfluidic devices, immunomagnetic cell separation for stem cell separation and isolation are the main focus in this paper. Further, devices like trans-coronary delivery, trans-endocardial delivery, intracoronary delivery devices for stem cell application to the heart are described. Devices for stem cell application to the brain, the spinal cord and other tissues are also explained. We highlighted scaffolds with incorporated stem cells and other encapsulation devices used to transplant stem cells. Current needs of devices for stem cells isolation, purification and delivery for drug discovery and cell therapy are discussed.

Endometrial stem cells provide a readily accessible source of adult stem cells suitable for various stem cell based therapies. The first essential step in the process of stem cell isolation, sorting and culture is tissue dissociation into single cell suspension. Tissue dissociation efficiency determines the number of resulting viable cells which in turn could affect the feasibility of the subsequent stem cell application. Microfluidic based platforms could provide several advantages over current conventional tissue dissociation techniques (e.g. mechanical or enzymatic or both) and help the optimization of the process towards a better reproducible cellular outcome. Objective: To develop a simple miniaturized platform for endometrial tissue dissociation which can be used to evaluate and optimize experimental conditions necessary for producing single cell suspension with minimal cell damage. Methods: A Poly-DiMethylSiloxane (PDMS) channel microfluidic platform was designed. The platform includes a tissue dissociation chamber where raw endometrial tissue was incubated. Trypsin was delivered through an inlet channel while another inlet enabled parallel delivery of a cell suspension medium and an outlet channel received the downstream dissociated endometrial cells. A cell size based filtration step is designed to trap large debris using two rows of posts. A viability stain is used to test the viability of cells produced at different enzyme-tissue contact durations. Conclusion: We present a simple microfluidics based platform for endometrial tissue dissociation that can be used for subsequent endometrial stem cell research applications and can be further applied to other tissues.

Mesenchymal stem cells (MSC) are increasingly being proposed as a therapeutic option for treatment of a variety of different diseases in human and veterinary medicine. Stem cells have been isolated from feline bone marrow, however, very few data exist about the morphology of these cells and no data were found about the morphometry of feline bone marrow-derived MSCs (BM-MSCs). The objectives of this study were the isolation, growth evaluation, differentiation potential and characterization of feline BM-MSCs by their morphological and morphometric characteristics. in vitro differentiation assays were conducted to confirm the multipotency of feline MSC, as assessed by their ability to differentiate into three cell lineages (osteoblasts, chondrocytes, and adipocytes). To evaluate morphological and morphometric characteristics the cells are maintained in culture. Cells were observed with light microscope, with association of dyes, and they were measured at 24, 48, 72 and 120h of culture (P1 and P3). The non-parametric ANOVA test for independent samples was performed and the means were compared by Tukey's test. On average, the number of mononuclear cells obtained was 12.29 (±6.05x10(6)) cells/mL of bone marrow. Morphologically, BM-MSCs were long and fusiforms, and squamous with abundant cytoplasm. In the morphometric study of the cells, it was observed a significant increase in average length of cells during the first passage. The cell lengths were 106.97±38.16µm and 177.91±71.61µm, respectively, at first and third passages (24 h). The cell widths were 30.79±16.75 µm and 40.18±20.46µm, respectively, at first and third passages (24 h).The nucleus length of the feline BM-MSCs at P1 increased from 16.28µm (24h) to 21.29µm (120h). However, at P3, the nucleus length was 26.35µm (24h) and 25.22µm (120h). This information could be important for future application and use of feline BM-MSCs. As células tronco mesenquimais são utilizadas na terapia de várias doenças na medicina humana e veterinária. As células tronco foram isoladas da medula óssea de gato, entretanto, existem poucos dados referentes a morfologia e não existem informações sobre a morfometria das células tronco isoladas da medula óssea. Os objetivos do presente estudo foram o isolamento, avaliação do crescimento, potencial de diferenciação e caracterização morfológica e morfométrica das células mesenquimais de gato isoladas de medula óssea. A diferenciação in vitro foi realizada para confirmar a multipotencialidade das células mesenquimais de gato (diferenciação em osteoblastos, condrócitos, adipócitos). As células mesenquimais foram mantidas em cultivo para avaliações morfológica e morfométrica. As células foram coradas e observadas em microscopia ótica. As mensurações foram realizadas com 24, 48, 72 e 120h de cultura (primeira e terceira passagens). O teste não paramétrico ANOVA foi utilizado e as médias foram comparadas pelo teste de Tukey. O número médio de células mononucleares obtido foi de 12,29 (±6,05x10(6)) células/mL de medula óssea. As células mesenquimais são longas e fusiformes, e escamosas com citoplasma abundante. No estudo morfométrico, observou-se aumento no comprimento médio das células durante a primeira passagem. As medidas de comprimento das células foram: 106,97±38,16µm e 177,91±71,61µm, respectivamente, na primeira e terceira passagens (24 horas). As medidas de largura das células foram: 30,79±16,75 µm e 40,18±20,46 µm, respectivamente, na primeira e terceira passagens (24 horas). O comprimento do núcleo na primeira passagem aumentou de 16,28µm (24h) para 21,29µm (120h) e na terceira passagem foi de 26,35µm (24h) para 25,22µm (120h). As informações são importantes para futuras aplicações e uso da célula mesenquimal de gato.

Regenerative medicine is an emerging field that is focused on the repair, replacement or regeneration of tissues and organs. It involves multiple disciplines dedicated to delivering different aspects of the regeneration process, including cell biology, material sciences and bioengineering. The development of tissue engineering strategies incorporating the use of autologous stem cells holds particular promise for overcoming insufficiencies from using cells from the patient's own diseased tissues and providing solutions for treatment of many disorders of the genitourinary tract. Many experimental projects have successfully utilized stem cells and several pilot studies in humans indicate the potential of stem cell therapy. However, the discipline is still young and further knowledge of both materials and stem cell biology is required before this promise can be realized through clinical application. This review examines the principles related to regenerative medicine and bioengineering focusing on the stem cell isolation expansion and clinical application. Analysis of current achievements will be reviewed alongside the challenges that remain to be addressed in considering the present and future perspectives of regenerative medicine applied to urology.

The Ly-6A and Ly-6E allelic genes encode the Sca-1 protein, which is one of the most widely used markers in haematopoietic stem cell isolation procedures. Identification of the specific gene regulatory elements that direct haematopoietic stem cell specific expression of Sca-1 is of current interest for purposes of stem cell manipulation. Both the Ly-6E and Ly-6A alleles have been examined for regions containing DNase I hypersensitive sites thought to be indicative of transcriptional regulatory elements. In these previous studies, the Ly-6E allele with its flanking regulatory sequences was cloned, and the region responsible for high-level γ-interferon (γ-IFN)-induced expression was localized to a 3′ distal sequence containing two strong DNase1 hypersensitive sites. Because the Ly-6A allele is thought to provide higher levels of expression in haematopoietic stem cells, we isolated over 25 kb of the Ly-6A gene and flanking regulatory regions. We show here that sequences analogous to those in the Ly-6E allele are responsible for high-level γ-IFN-induced expression in vitro. Furthermore, we show that this 3′ distal Ly-6A fragment directs high-level γ-IFN-induced expression from a heterologous promoter, suggesting that it is a potent enhancer that could be useful for expression in haematopoietic stem cells.

We developed a combined methodological approach to enrich and to proliferate in vitro fetal CD34+ stem progenitor cells. Using a magnetic cell-sorting technique, CD34+ cells from pregnant women at the early-second trimester were isolated and enriched and compared to those isolated from blood of nonpregnant women. The number and frequency of CD34+ cells were significantly higher (p0.001) in the pregnant women. Unenriched peripheral blood mononuclear cells (PBMC) and enriched CD34+ cells were cultured in a methylcellulose system to evaluate the cloning potential of progenitor cells. After culture, the numbers of burst-forming units erythroid/colony-forming units erythroid (BFU-E/CFU-E) and colony-forming units granulocyte-macrophage (CFU-GM) colonies were increased by 33 and 16 times, respectively. Finally, to distinguish between fetal and maternal cells, four cases of cultured cells were hybridized with specific probes for X and Y chromosomes and two cases with a specific probe for chromosome 21. In normal pregnancies, we identified a high number of male fetal cells and an elevated fetal/maternal ratio. When we analyzed blood samples from pregnancies with trisomic fetuses, we scored a high ratio of trisomic cells respect to maternal cells that was significantly different from the ratio of pregnancies with normal fetuses. Our results demonstrate fetal progenitor cells may be cultured and detected successfully with an appropriate combined methodological approach, which may significantly increase the feasibility of noninvasive prenatal diagnosis.

Recently we and two other groups have shown that human spermatogonial stem cells (SSCs) have the potential to become pluripotent in vitro in defined culture conditions and to differentiate into cells of the three embryonic germ layers. This discovery could open new avenues for autologous cell-based therapy in degenerative diseases, bypassing the ethical and immunological problems related to the human embryonic stem cells. In addition, human SSCs could be used to treat infertility in cancer survival children. However, in order to reprogram SSCs into pluripotency, or to preserve them for repopulation of infertile testes, the first and limiting step is to have access to a highly purified human SSC population that could be multiplied and efficiently cultured in vitro maintaining their molecular and cellular characteristics. Although various studies have attempted to identify molecular markers of human SSCs, to date there is still limited information related to the specific markers that could be used for their isolation and optimized purification that allows long-term in vitro culture of isolated human SSCs. Here using SSEA-4 as an optimal marker for isolation of a subpopulation of SSCs, we show that SSEA-4 positive cells express the highest level of SSC genes compared to other subpopulations isolated with different markers, and can be maintained in culture for over 14 passages which we were unable to obtain with other SSCs markers including GPR125 and ITGA6. In addition, we have established a new technology for cell sorting and long-term culture of human SSC-SSEA-4 positive cells that maximizes the purity and viability of the sorted cells. Our findings are crucial and could be used for the most efficient isolation, purification and long-term culture of SSCs for clinical applications in regenerative medicine, or for preparation of human SSCs for autologous treatment of infertility in cancer survival children.

Annually, more than 200,000 elective liposuction procedures are performed in the United States and over a million worldwide. The ease of harvest and abundance make human adipose-derived stromal/stem cells (hASCs) isolated from lipoaspirates an attractive, readily available source of adult stem cells that have become increasingly popular for use in many studies. Here, we describe common methods for hASC culture, preservation, and osteogenic differentiation. We introduce methods of ceramic, polymer, and composite scaffold synthesis with a description of morphological, chemical, and mechanical characterization techniques. Techniques for scaffold loading are compared, and methods for determining cell loading efficiency and proliferation are described. Finally, we provide both qualitative and quantitative techniques for in vitro assessment of hASC osteogenic differentiation.

Subcutaneous adipose tissue is ubiquitous and easily accessible in large quantities with a minimal invasive procedure by liposuction aspiration, qualifying it as a good source for stem cell isolation. Adipose-derived mesenchymal stromal/stem cells (ASCs) can easily be isolated by a relatively simple method, yielding a high amount of stem cells, which is essential for stem cell-based therapies and tissue engineering. Several studies have provided evidence that ASCs in situ reside in the wall surrounding the vasculature, and that blood vessels in virtually all organs and tissues harbour ubiquitous (mesenchymal) stem cells in their perivascular niche. Nevertheless, the exact localization of ASCs in native adipose tissue is still under debate. ASCs are isolated by their capacity to adhere to cell culture plastic and are maintained in culture for up to 10–15 passages. Cultured cells are characterized by their expression of a panel of characteristic markers and their capacity to differentiate into cells from meso-, ecto- and endodermal lineages. ASCs possess a high plasticity and differentiate into various cell types, including endothelial cells in vitro and in vivo. This review summarizes and discusses the current knowledge about ASCs and their differentiation potential into endothelial cells in vitro and in vivo.

Recent advances in harvesting techniques and radiological imaging protocols have resulted in fat graft breast augmentation becoming an alternative to implant augmentation. Adipose-derived stem cells are well known for their regenerative and therapeutic properties and have the ability to differentiate along multiple lineage pathways. The subcutaneous adipose tissue is the largest reservoir of adipose-derived stem cells (ADSCs). Liposuction is thus a convenient method of obtaining large amounts of ADSCs and eliminates the need for tissue culturing. The authors discuss stem cell-enhanced fat graft breast augmentation and describe the technique for adipose-derived stem cell isolation for fat grafting.

Spermatogonial stem cells (SSCs) comprise a small population of germ cells that have self-renewal potential. However, studies on SSCs are hampered by the lack of SSC-specific markers. Although the cryptorchid operation is often used to obtain an enriched SSC population by destroying differentiating germ cells using high body temperature, SSCs in cryptorchid testes have different biological characteristics than those in a normal environment. Therefore, it is necessary to develop new methods for SSC selection. In this study, we report a method of isolating SSCs from wild-type mouse testes based on their functional characteristics using aldehyde dehydrogenase (ALDH) activity levels, which have been successfully used for stem cell isolation from many self-renewing tissues. Testis cells selected using CD9 or CDH1, both of which are expressed by SSCs, exhibit ALDH activity in flow cytometric analyses. However, spermatogonial transplantation revealed that SSCs do not show ALDH activity, whereas somatic ...

Autologous therapy via stem cell-based tissue regeneration is an aim to rebuild natural teeth. One option is the use of adult stem cells from the dental pulp (DPSCs), which have been shown to differentiate into several types of tissue in vitro and in vivo, especially into tooth-like structures. DPSCs are mainly isolated from the dental pulp of third molars routinely extracted for orthodontic reasons. Due to the extraction of third molars at various phases of life, DPSCs are isolated at different developmental stages of the tooth.The present study addressed the question whether DPSCs from patients of different ages were similar in their growth characteristics with respect to the stage of tooth development. Therefore DPSCs from third molars of 12-30 year-old patients were extracted, and growth characteristics, e.g. doubling time and maximal cell division potential were analysed. In addition, pulp and hard dental material weight were recorded.Irrespective of the age of patients almost all isolated cells reached 40-60 generations with no correlation between maximal cell division potential and patient age. Cells from patients22 years showed a significantly faster doubling time than the cells from patients ≥22 years.The age of patients at the time of stem cell isolation is not a crucial factor concerning maximal cell division potential, but does have an impact on the doubling time. However, differences in individuals regarding growth characteristics were more pronounced than age-dependent differences.

Mammalian adipose tissue derived stem cells (AT-SC) have a tremendous potential in regenerative medicine for tissue engineering and somatic nuclear transfer (SNT). The isolation methods of human and bovine adipose tissue derived stem cells are compared in this paper to determine the feasibility and optimum method of isolation. The optimum isolation method will reduce the processing time, efforts and money as isolation is the first crucial and important step in stem cells research. Human abdominal subcutaneous adipose tissue and bovine abdominal subcutaneous adipose tissue are digested in three collagenase type 1 concentration 0.075%, 0.3% and 0.6% agitated at 1 h and 2 h under 37 °C in 5% CO2 incubator. The cultures are then morphologically characterised. Human adipose tissue stem cells are found to be best isolated using abdominal subcutaneous depot, using 0.075% collagenase type 1 agitated at 1 h under 37 °C in CO2 incubator. While bovine adipose tissue derived stem cells are best isolated using abdominal subcutaneous depot, using 0.6% collagenase type 1 agitated at 2 h under 37 °C in CO2 incubator.

Dental pulp stem cells (DPSCs) have therapeutic potential for dentin and dental pulp regeneration. For regenerative approaches to gain clinical acceptance, protocols are needed to determine feasible ways to store teeth, isolate DPSCs, and expand them to clinical scale numbers.In this study, 32 third molars were obtained from patients and immediately placed in saline or tissue culture medium followed by overnight storage at 4°C or immediate isolation of DPSCs. Upon isolation, cells were expanded in medium containing either fetal bovine serum (FBS) or human serum (HS). Cell proliferation (population doubling time [PDT]), cell surface marker expression, and multipotency were compared between DPSCs in FBS and DPSCs in HS.The time frame of storage and storage medium did not affect the ability to isolate DPSCs. However, using HS instead of FBS in the initial isolation of DPSCs significantly decreased (P.01) the isolation success rate from 89% (FBS) to 23% (HS). Yet, incorporating fibronectin in the DPSC initial isolation (using HS) significantly (P.01) increased the isolation success rate to 83%. Interestingly, it was found that the proliferation rate was significantly (P.05) higher for DPSCs in HS (PDT = 1.59 ± 0.46) than that for DPSCs in FBS (PDT = 2.84 ± 2.5). Finally, there was no difference in the expression of CD73, CD90, CD105, or multipotency (as measured by osteogenic, adipogenic, and chondrogenic differentiation) between DPSCs in FBS and DPSCs in HS.These findings show a clinically feasible method of storing third molars for the isolation of DPSCs. Additionally, DPSCs can be isolated and expanded to clinical scale numbers in media devoid of FBS and still maintain their phenotypic properties.

Variability in harvest and processing technique may impact the success of fat grafting. This study compared properties of fat grafts produced by differing methods and assessed volume retention of the grafted tissue in a nude mouse model.In phase I, fat was harvested by either suction-assisted lipoaspiration or ultrasound-assisted lipoaspiration and then filtered using two different pore sizes. Graft material was analyzed for average parcel size; relative oil, fat, and aqueous fractions; and stromal vascular fraction yield. Filtrands and filtrates were injected into athymic nude mice. In phase II, lipoaspirate harvested by suction-assisted lipoaspiration only was processed by centrifugation, cotton gauze rolling, or filtration, and then studied in a similar manner.Fat harvested by ultrasound- and suction-assisted lipoaspiration had comparable stromal vascular fraction counts and graft retention in vivo. Ultrasound-assisted lipoaspiration released only slightly more oil than suction-assisted lipoaspiration; filtering with either 500- or 800-µm pore size effectively removed fluid and oil. Centrifugation, cotton-gauze rolling, and filtration also effectively removed fluid and oil. In vivo graft retention and stromal vascular fraction yield was highest with the cotton gauze method. Histologic analysis of all explants showed intact adipose tissue.Ultrasound- and suction-assisted lipoaspiration yielded similar retention of fat grafts in a xenograft model. Processing with cotton gauze rolling may be best suited for grafting cosmetically sensitive areas of the body in which optimal retention is critical and lower total graft volumes are needed. Filtration and centrifugation both effectively removed fluid fractions and resulted in comparable graft retention, and are more feasible when larger volumes are required.

Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughout the lifetime of the animal. While experimenting with staining of murine bone marrow cells with the vital dye, Hoechst 33342, we discovered that display of Hoechst fluorescence simultaneously at two emission wavelengths revealed a small and distinct subset of whole bone marrow cells that had phenotypic markers of multipotential HSC. These cells were shown in competitive repopulation experiments to contain the vast majority of HSC activity from murine bone marrow and to be enriched at least 1,000-fold for in vivo reconstitution activity. Further, these Hoechst-stained side population (SP) cells were shown to protect recipients from lethal irradiation at low cell doses, and to contribute to both lymphoid and myeloid lineages. The formation of the Hoechst SP profile was blocked when staining was performed in the presence of verapamil, indicating that the distinctly low staining pattern of the SP cells is due to a multidrug resistance protein (mdr) or mdr-like mediated efflux of the dye from HSC. The ability to block the Hoechst efflux activity also allowed us to use Hoechst to determine the DNA content of the SP cells. Between 1 and 3% of the HSC were shown to be in S-G2M. This also enabled the purification of the G0-G1 and S-G2M HSC had a reconstitution capacity equivalent to quiescent stem cells. These findings have implications for models of hematopoietic cell development and for the development of genetic therapies for diseases involving hematopoietic cells.

Bulge stem cells reside in the lowest permanent portion of hair follicles and are responsible for the renewal of these follicles along with the repair of the epidermis during wound healing. These cells are identified by surface expression of CD34 and the α6-integrin. When CD34 and α6 double-positive cells are isolated and implanted into murine skin, they give rise to epidermis and hair follicle structures. The current gold standard for isolation of these stem cells is fluorescence-activated cell sorting (FACS) based on cell surface markers. Here, we describe an alternative method for CD34 bulge stem cell isolation: a microfluidic platform that captures stem cells based on cell surface markers. This method is relatively fast, requiring 30 min of time from direct introduction of murine skin tissue digestate into a two-stage microfluidic device to one-pass elution of CD34(+) enriched cells with a purity of 55.8% ± 5.1%. The recovered cells remain viable and formed colonies with characteristic morphologies. When grown in culture, enriched cells contain a larger α6(+) population than un-enriched cells.

The adult heart has been traditionally considered to be a postmitotic organ, but recent evidence, from animal and human studies, has demonstrated a limited capacity for cell renewal in the organ. This regenerative capacity has been attributed to the presence of stem cells in the adult heart, and distinct stem cell populations have been described in the literature. In this chapter we discuss the evidence for self-renewal of cardiomyocytes and describe the various progenitor/stem cell types that have been proposed to reside in the heart, based on the expression of surface markers or on functional assays. Based on the limited regenerative capacity of the heart, it is unlikely that the organ harbors distinct populations of progenitor/stem cells, and we propose that identifying a set of markers and functional properties would provide a more robust method for progenitor/stem cell isolation, characterization, and expansion.

Primitive stem cells have been discovered within the blood of adult mammals such as rodents, porcines, and humans. The current study addressed the issue of primitive stem cells in the blood of adult equines. Blood withdrawal by venipuncture from adult equines was performed following the guidelines of Fort Valley State University IACUC. Ten horses were used in this study: one Danish Oldenburg, three Standard breds and six Quarter Horses with age ranges of 5 – 20 years. The blood was processed for stem cell isolation and counting. All horses examined were noted to have circulating levels of primitive stem cells in their blood. Standard breds showed an increase of stem cell number with increasing age of the animals. In contrast, Quarter Horses showed an increase in stem cell number that paralleled an increase in the level of stress to the animal, regardless of age. All horses showed an increase in stem cell number in their blood after moderate exercise (10 minutes of cantering) and at time periods after ingestion of Aphanizomenon flos-aquae . These studies demonstrate the existence of primitive stem cells within adult equine blood. Larger sample sizes are needed to determine the significance of the effects of age, stress, trauma and ingested compounds on the number of circulating primitive adult stem cells in the blood of adult horses. Further studies are also needed to assess the applicability of using circulating primitive stem cells for the restoration and/or repair of tissues in the adult equine damaged by trauma or disease.

The transition from single cell species to multicellular organisms led to organ and tissue development. The development and maintenance of organs/tissues in multicellular organisms has resulted in the emergence of specialized cells that self-renew as well as differentiate; these rare cells are stem cells. Following embryonic development, most of our tissues and organs are continuously regenerated from tissue/organ specific stem cells. The principal property that distinguishes such stem cells from their daughter cells is self-renewal; when stem cells divide they give rise to stem cells (by self-renewal) and progenitors (by differentiation). In most tissues only the primitive stem cells self-renew. Even within a particular tissue, stem cells may be diverse, and subject to stem cell competition for their niche. We have documented both germline stem cell and somatic stem cell competition in species as diverse as colonial protochordates and humans. Stem cell isolation and transplantation is the basis for regenerative medicine. Self-renewal is dangerous, and therefore strictly regulated. Poorly regulated self-renewal can lead to the genesis of cancer stem cells, the only self-renewing cells in the cancerous tumor. The Weissman lab has followed the progression from hematopoietic stem cells (HSCs) to myelogenous leukemias. They have found that the developing cancer clones progress at the stage of HSCs, until they become fully malignant. At this point, the “leukemia” stem cell is at a stage of a downstream oligolineage or multilineage progenitor that has evaded programmed cell death and programmed cell removal, while acquiring or keeping self-renewal. In the case of chronic myeloid leukemia, bcr-abl+ HSC clones outcompete normal HSCs in the chronic phase. The transition from the chronic phase to myeloid blast crisis results in the leukemia stem cells appearing in the granulocyte-macrophage progenitor (GMP) stage. While there are many ways to defeat programmed cell death and senescence, there appears to be one dominant method to avoid programmed cell removal—the expression of the cell surface “don't eat me” protein CD47, the ligand for macrophage SIRPα. All cancers tested express CD47 to overcome expression of “eat me” signals such as calreticulin. Antibodies that block the CD47-SIRPα interaction enable phagocytosis and killing of the tumor cells in vitro and in vivo. All tested human solid tumors and lymphomas/leukemias/myelomas express CD47 and are susceptible to phagocytosis in the presence of anti-CD47 blocking antibodies. Citation Format: Irving L. Weissman. Normal and neoplastic stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr SY08-02.

Different approaches for the isolation of stem/progenitor cells have been reported, including stem cell selection in stringent culture conditions. We evaluated the possibility of isolating human progenitor cells from surgical specimens preserved by under vacuum sealing and cooling, a clinical practice approached by several hospitals as alternative to formalin. Renal tissue samples (n = 20) maintained under vacuum from 6 to 48 h at 4°C were used to isolate human renal CD133(+) progenitor cells. We obtained CD133(+) progenitors from unsorted cells derived from disaggregated tissues from each sample. Phenotypic characterization as well as in vitro and in vivo differentiation of the obtained CD133(+) lines showed results comparable with sorted CD133(+) cells obtained from fresh tissue. These results indicate that the process of sealing under vacuum and cooling appears as a suitable tissue treatment to isolate hypoxia resistant cells, such as human stem/progenitor cells, and that this procedure can be exploited to render the extraction of stem cells from human samples more practical and feasible.

Large body surface area burns pose significant therapeutic challenges. Clinically, the extent and depth of burn injury may mandate the use of allograft for temporary wound coverage while autografts are serially harvested from the same donor areas. The paucity of donor sites in patients with burns involving large surface areas highlights the need for better skin substitutes that can achieve early and complete coverage and retain normal skin durability with minimal donor requirements. We have isolated autologous stem cells from the adipose layer of surgically debrided burned skin (dsASCs), using a point-of-care stem cell isolation device. These cells, in a collagen—polyethylene glycol fibrin-based bilayer hydrogel, differentiate into an epithelial layer, a vascularized dermal layer, and a hypodermal layer.All-trans-retinoicacid and fenofibrate were used to differentiate dsASCs into epithelial-like cells. Immunocytochemical analysis showed a matrix- and time-dependent change in the expression of stromal, vascular, and epithelial cell markers. These results indicate that stem cells isolated from debrided skin can be used as a single autologous cell source to develop a vascularized skin construct without culture expansion or addition of exogenous growth factors. This technique may provide an alternative approach for cutaneous coverage after extensive burn injuries.

Novel therapy with mesenchymal stem cells from bone marrow, adipose tissue, or other sources has raised high hopes for treatment of a variety of diseases. For plastic and reconstructive surgery, first pilot studies and clinical trials using stem cells for treatment of chronic wounds, radiation injury, or soft tissue augmentation have furnished encouraging results compared with the limitations of standard therapy, for example autologous fat grafting. Further research must be conducted to reveal the complex physiological interactions between activated stem cells and the host environment. Long-term effects and safety aspects of these novel treatment options also require randomized controlled studies. For future clinical applications, guidelines and standardized procedures for stem cell isolation and preparation, and techniques for application must be established.

International audience; BACKGROUND:Adipose stem cells have gained great interest in plastic and reconstructive surgery with their ability to improve engraftment after fat transfer for soft tissue filling. It is therefore essential to know the effect of the drugs commonly used during the lipoaspiration procedure, such as lidocaine and adrenaline. Indeed, these drugs are infiltrated at the fat donor site for local anesthesia and for reduction of bleeding. This study analyzed the effects of these drugs on the viability of adipose-derived stem cells and on their inflammatory status.METHODS:Adipose-derived stem cells from lipoaspirates were grown in culture before being treated with different clinical doses of lidocaine at different times of exposure (1-24 h), and with adrenaline (1 μg/mL). Cytotoxicity was measured by lactate dehydrogenase assay and by flow cytometry with annexin V/propidium iodide staining. In parallel, the secretion of the proinflammatory cytokines tumor necrosis factor-alpha (TNFα), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1) was tested by enzyme-linked immunoassay.RESULTS:Lidocaine affected cell viability after 24 h, even when the cells were exposed for only 1 or 2 h. Apoptosis was not involved in lidocaine cytotoxicity. Regarding inflammation, no TNFα was produced, and lidocaine decreased the levels of IL-6 and MCP-1 in a dose-dependent manner. In contrast, adrenaline did not influence cell viability or cytokine secretions.CONCLUSIONS:Adipose tissue should be handled appropriately to remove lidocaine and adrenaline, with such procedures as washing and centrifugation. This study provides new insights into the use of lidocaine and adrenaline for fat transfer or stem cell isolation from lipoaspirates.LEVEL OF EVIDENCE II:This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Treatments for neurodegenerative diseases have little impact on the long-term patient health. However, cellular transplants of neuroblasts derived from the aborted embryonic brain tissue in animal models of neurodegenerative disorders and in patients have demonstrated survival and functionality in the brain. However, ethical and functional problems due to the use of this fetal tissue stopped most of the clinical trials. Therefore, new cell sources were needed, and scientists focused on neural (NSCs) and mesenchymal stem cells (MSCs). When transplanted in the brain of animals with Parkinson's or Huntington's disease, NSCs and MSCs were able to induce partial functional recovery by promoting neuroprotection and immunomodulation. MSCs are more readily accessible than NSCs due to sources such as the bone marrow. However, MSCs are not capable of differentiating into neurons in vivo where NSCs are. Thus, transplantation of NSCs and MSCs is interesting for brain regenerative medicine. In this chapter, we detail the methods for NSCs and MSCs isolation as well as the transplantation procedures used to treat rodent models of neurodegenerative damage.

Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological sciences, such as tissue engineering, regenerative medicine, rare cell and stem cell isolation, proteomic/genomic research, and clonal/population analyses.

The amniotic fluid or liquor amnii, was first isolated and studied during the beginning of the 20th century (Brace et al., 1989). More recently, in the 1960s and 1970s there was an increased interest in characterization and culture of the cells contained in the amniotic fluid (Huisjes HJ, 1970; Marchant GS, 1971). Nevertheless, most all of these studies were directed at using amniotic fluid, and the cells contained within, for determining the health of the fetus during development, or to provide a general characterization of the amniotic fluid. Although the discovery of stem cells, in particular bone marrow stem cells, occurred in the 1960’s, it was not until recently that the possibility of isolating stem cells from the amniotic fluid was investigated. Amniotic fluid stem cell isolation and characterization is therefore fairly recent, dating back to the early 1990’s (Torricelli et al., 1993). The study of amniotic fluid-derived stem cells (AFDSCs) has captured the attention of researchers and clinicians for several reasons. First, AFDSCs can be collected during amniocentesis and isolated from material that would be otherwise discarded. Therefore, their use is not subject to the ethical debate that surrounds the use of embryonic stem cells. Second, like other fetal derived stem cells, storage of AFDSCs is easy and achieved at minimal costs. AFDSC populations can be easily expanded, and have shown the capability of being stored over long periods of time with no adverse effects (Da Sacco et al., 2010). Furthermore, the “banking” of AFDSCs from developing fetuses, may guarantee a source of stem cells with a matching immune profile to that of the recipient. Most importantly, the extensive characterization of a specific subset of AFDSCs positive for the marker c-Kit+ (De Coppi et al., 2007), have displayed no tumor formation following transplant into an animal model, even after several months. These cells, known simply as amniotic fluid stem cells (AFSC) have been at the forefront of AFDSC research and will be discussed in depth later. Finally, as a source of stem cells collected before birth AFDSCs may become an invaluable source of stem cells for direct treatment of various genetic disorders treatable in utero (Turner CG et al., 2009). The potential applications and implications of AFDSCs in regenerative medicine and therapeutic treatments are significant, however; AFDSCs research is still in its infancy and much work is required to properly characterize AFDSCs and determine their effectiveness. In this chapter, we describe the different AFDSCs that have been isolated to date, list their characteristics, and provide an overview of the different organs in which AFDSCs have been used in vitro or in vivo to develop this stem cell population into a viable therapeutic strategy.

Introduction Ligament and tendon are prone to degeneration through ageing and injury and current therapies are largely ineffective. The identification of a cell population within tendon with stem cell-like characteristics (Bi, 2007) holds potential for regeneration of tendon and ligament. Tendon stem cells differentiate into tenocytes (Zhang, 2010); the predominant cell type within tendon, responsible for producing extracellular matrix (ECM). The local stem cell environment (niche) is vital for stem cell maintenance and function in many tissues, and tenascin C in particular has been shown to play an important role within stem cell niches (Garcion, 2004). Tendon and ligament are composed of fascicles and interfascicular matrix (IFM) which vary considerably in composition providing definitive niches within the tissue. This study aims to characterise ECM components of the stem cell niche in equine tendon and canine ligament, which are prone to age-related degeneration. The goal of this research is to produce an in vitro environment for stem cells which mimics the stem cell niche, for treatment of tendon and ligament disease. Methods Putative stem cells were isolated from equine superficial digital flexor tendon (SDFT) and canine anterior cruciate ligament (ACL) by low-density plating and differential adhesion to plastic and fibronectin substrates. Cells were analysed by flow cytometry using antibodies to mesenchymal stem cell markers CD90, CD73 and CD105, as well as qRT-PCR for stem cell and tenogenic markers. ECM components of the fibroblast and stem cell niche were analysed using radioisotope labelling. Cells were labelled with 14 C-labelled amino acids to specifically label newly synthesised collagenous (proline) and non-collagenous (lysine/arginine) ECM, prior to extraction of ECM. Immuno-histochemistry and histology were conducted to analyse the structure and composition of SDFT. Results Tendon and ligament cells formed colonies after low-density plating, however only ligament cells formed colonies after differential adhesion to fibronectin. A subpopulation of tendon cells expressed CD90 in both freshly isolated cells and putative stem cells, but were CD105 and CD73 negative. Putative tendon stem cells, isolated by differential fibronectin adhesion did not exhibit increased expression of stem cell markers when compared with tenocytes. However there was a significant increase in expression of stem cell markers in putative ligament stem cells compared with ligamentocytes. Tenocytes and putative tendon stem cells (isolated by low-density plating) labelled with 14 C-labelled amino acids both displayed similar labelling profiles. Histological analysis of SDFT tissue highlighted the varied structure and composition of tendon, with tenascin C expression confined to IFM (see Figure.1). Conclusion The absence of stem cell marker expression in putative stem cell populations indicates that further testing of stem cell isolation procedures is required. Published techniques for tendon stem cell isolation in humans and other mammals do not appear to be effective for isolation of equine tendon stem cells. Alternatively it is possible that the equine tendon cell population consists of a heterogenous mixture of cells at different stages of differentiation. We are currently optimising stem cell isolation techniques and conducting tri-lineage differentiation assays. Tendon stem cells in other species show tri-lineage differentiation, however it is possible that equine tendon stem cells may be restricted to tenogenic differentiation. Future experiments aim to identify ECM components of the stem cell niche by mass spectrometry and microarray comparison of tendon and ligament tissue, stem cells and fibroblasts. References Bi et al . Nat Med. 2007;13: 1219–1227 Garcion et al . Development. 131:3423–3432 Zhang et al . Am J Sport Med. 38:2477–2486

Mammalian adipose tissue derived stem cells have a tremendous potential in regenerative medicine for tissue engineering and somatic nuclear transfer (SNT). The isolation methods of human and bovine adipose tissue derived stem cells are compared in this paper to determine the feasibility and optimum method of isolation. The optimum isolation method will reduce the processing time, efforts and money as isolation is the first crucial and important step in stem cells research. Human abdominal subcutaneous adipose tissue and bovine abdominal subcutaneous adipose tissue are digested in three collagenase type 1 concentration 0.075%, 0.3% and 0.6% agitated at 1 hour and 2 hours under 37°C in 5% CO 2 incubator. The cultures are then morphologically characterised. Human adipose tissue stem cells are found to be best isolated using abdominal subcutaneous depot, using 0.075% collagenase type 1 agitated at 1 hours under 37°C in CO 2 incubator. While bovine adipose tissue derived stem cells are best isolated using abdominal subcutaneous depot, using 0.6% collagenase type 1 agitated at 2 hours under 37°C in CO 2 incubator.

BACKGROUND AIMS: Bone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance. METHODS: BM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential. RESULTS: No differences were noticed in cell recovery and viability between the groups. Fluorescence-activated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45(+) fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage. CONCLUSIONS: Both GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications.

It has been postulated that brain tumor stem cells (TSCs) may be the population of cells responsible for the maintenance and recurrence of glioblastoma multiforme (GBM). The purpose of this study was to optimize a reproducible protocol for generating TSCs for their subsequent transfection or transduction. Patient GBMs were enzymatically and mechanically dissociated and tumor spheres were resuspended in appropriate media and analyzed to ensure they met stem cell criteria. These cells were then transfected with a plasmid or transduced with a viral vector to introduce a previously absent gene and then allowed to form tumor spheres. Tumor spheres were generated from patient GBMs without contamination. These cells met stringent criteria as stem cells, including multipotentiality and self-renewal. High efficiency transfection and transduction of tumor spheres was possible, even at the core of the sphere. This allowed for the introduction of new genes to the TSCs, as evidenced by fluorescent microscopy and Western blot analysis. This study is a guide to optimize the generation of patient derived GBM tumor spheres without RBC and dead cell contamination. GBM TSCs within tumor spheres can easily be transfected with plasmids or transduced with a virus. This is important from a therapeutic perspective if gene replacement is to be successful in replacing genes lost in GBM progression or to knock down or silence genes that are over-expressed in malignant brain tumors.

International audience; The characterisation of normal stem cells and cancer stem cells uses the same paradigm. These cells are isolated by a fluorescence-activated cell sorting step and their stemness is assayed following implantation into animals. However, differences exist between these two kinds of stem cells. Therefore, the translation of the experimental procedures used for normal stem cell isolation into the research field of cancer stem cells is a potential source of artefacts. In addition, normal stem cell therapy has the objective of regenerating a tissue, while cancer stem cell-centred therapy seeks the destruction of the cancer tissue. Taking these differences into account is critical for anticipating problems that might arise in cancer stem cell-centred therapy and for upgrading the cancer stem cell paradigm accordingly.