Characterization Of Adult Stem Cells Research Articles

Neural, adipocyte and hepatic differentiation potential of primary and secondary hair follicle stem cells isolated from Arbas Cashmere goats

BackgroundArbas Cashmere goats are excellent domestic breeds with high yields of wool and cashmere. Their wool and cashmere can bring huge benefits to the livestock industry. Our studies intend to more fully understand the biological characteristics of hair follicle stem cells (HFSCs) in order to further explore the mechanisms of wool and cashmere regular regeneration. And they have been increasingly considered as promising multipotent cells in regenerative medicine because of their capacity to self-renew and differentiate. However, many aspects of the specific growth characteristics and differentiation ability of HFSCs remain unknown. This study aimed to further explore the growth characteristics and pluripotency of primary hair follicle stem cells (PHFSCs) and secondary hair follicle stem cells (SHFCs).ResultsWe obtained PHFSCs and SHFSCs from Arbas Cashmere goats using combined isolation and purification methods. The proliferation and vitality of the two types of HFSCs, as well as the growth patterns, were examined. HFSC-specific markers and genes related to pluripotency, were subsequently identified. The PHFSCs and SHFSCs of Arbas Cashmere goat have a typical cobblestone morphology. Moreover, the PHFSCs and SHFSCs express HFSC surface markers, including CD34, K14, K15, K19 and LGR5. We also identified pluripotency-associated gene expression, including SOX2, OCT4 and SOX9, in PHFSCs and SHFSCs. Finally, PHFSCs and SHFSCs displayed multipotent abilities. PHFSCs and SHFSCs can be directed to differentiate into adipocyte-like, neural-like, and hepatocyte-like cells.ConclusionsIn conclusion, this study confirmed that the biological characteristics and differentiation potential of PHFSCs and SHFSCs from Arbas Cashmere goats. These findings broaden and refine our knowledge of types and characteristics of adult stem cells.

Metabolic determination of cell fate through selective inheritance of mitochondria.

Metabolic characteristics of adult stem cells are distinct from their differentiated progeny, and cellular metabolism is emerging as a potential driver of cell fate conversions 1–4 . How these metabolic features are established remains unclear. Here, we identified inherited metabolism imposed by functionally distinct mitochondrial age-classes as a fate determinant in asymmetric division of epithelial stem-like cells. While chronologically old mitochondria support oxidative respiration, the electron transport chain of the new organelles is proteomically immature and they respire less. Upon cell division, selectively segregated mitochondrial age-classes elicit a metabolic bias in progeny cells, with oxidative energy metabolism promoting differentiation in cells inheriting old mitochondria. Cells inheriting newly synthesised mitochondria with low levels of Rieske iron-sulfur polypeptide 1 have a higher pentose phosphate pathway activity, which promotes de-novo purine biosynthesis and redox balance, and is required to maintain stemness during early fate determination after division. Our results demonstrate that fate decisions are susceptible to intrinsic metabolic bias imposed by selectively inherited mitochondria.

Spermatogonial stem cells: updates from specification to clinical relevance.

Human spermatogonia are target for exploration of adult stem cell characteristics and potential source for the development of therapeutic applications. Almost 50 years ago, Yves Clermont stated with regard to the nature of the true stem cells: 'there is the possibility that other classes of spermatogonia exist beside the three classes (Adark, Apale and type B)…; …we still know too little about the human spermatogonial stem cells'… This review seeks to provide current knowledge, focusing on different aspects of human spermatogonia, and novel information based on species comparisons with regard to the adaptation of their proliferative potential. Moreover, the objective is to provide an update on the state of the art concerning the potential use of human spermatogonia for clinical applications. Germ cell specification mechanisms and epigenetic as well as transcriptional features of primordial germ cells (PGC) and adult spermatogonia at the single-cell level are reviewed. Studies on single-cell analyses have been included as they provide hitherto unequaled resolution of the transcriptional profiles of unselected human testicular cells and, thereby, new insights into the molecular aspects of germ cell differentiation. Datasets on models of spermatogonial expansion were identified and spermatogonial turnover and lifetime sperm production rates in various species were calculated, based exclusively on studies employing the optical dissector approach. Finally, the state of the art concerning causes of impaired spermatogonial function and fertility preservation were comprehensively reviewed. RNA sequencing data from PGC and spermatogonia indicate that transcriptional heterogeneity is a feature of germ cells prior to differentiation. Based on these data as well as lineage-tracing studies it is now debated whether spermatogonia are a rather plastic population of undifferentiated germ cells with the stem cell niche being the regulatory unit for cell fate decisions. Based on our novel calculations we suggest that spermatogonia are adapted to the individual reproductive lifespan and that the life-long sperm output from a spermatogonium is balanced against the duration of a generation. Thereby, the risk of jeopardizing genome integrity is balanced against a maximized sperm output. With reference to Yves Clermont's statement, and based on recent datasets, we suggest that the question that needs to be answered is: 'Is there a true stem cell?' or better 'Is there a population of various cells with distinct features serving as a stem cell pool?'. This review provides an update including novel views on various aspects of spermatogonial biology (from embryonic to adult stages). We consider this review relevant for all research scientists and clinicians dealing with fertility, spermatogenesis and fertility preservation.

Adipose tissue-derived stromal cells (ADSC) express oligodendrocyte and myelin markers, but they do not function as oligodendrocytes.

Mesenchymal cells cultured from the vasculo-stromal fraction of adipose tissue (ADSC) show adult stem cell characteristics and several groups have claimed generating neural cells from them. However, we have observed that many markers commonly used for the identification of neural cells are spontaneously expressed by ADSC in culture. In the present study, we have examined the expression of characteristic oligodendrocyte molecules in cultured ADSC, aiming to test if myelinating cells could be generated from accessible non-neural adult tissues. In basal growth conditions, rat ADSC spontaneously expressed CNPase, MBP, MOG, protein zero, GAP43, Sox10, and Olig2, as shown by immunocytrochemistry and western blot. A small population of cultured ADSC expressed membrane galactocerebroside (O1 antibody), but no cell stained with O4 antibody. RT-PCR analyses showed the expression of CNPase, MBP, DM20, and low levels of Olig2, Sox10, and Sox2 mRNA by rat ADSC. When rat ADSC were treated with combinations of factors commonly used in neural-inducing media (retinoic acid, dbcAMP, EGF, basic FGF, NT3, and/or PDGF), the number of O1-positive cells changed, but in no case, mRNA expression of Sox10 and Olig2 transcription factors approached CNS oligodendrocyte levels. In co-culture with rat dorsal root ganglion neurons, no sign of axonal myelination by rat ADSC was observed. These studies show that the expression of oligodendrocyte traits by cultured ADSC is not a proof of functional competence as oligodendroglia and suggest that in culture conditions, ADSC acquire intermediate, uncommitted phenotypes.

Proliferation and Differentiation of Human Hair Follicle Stem Cells on Chitosan-Skin Engineered Template in Vitro

Hair follicles repeatedly regress and reconstitute themselves, suggesting the presence of intrinsic tissue stem cells. Among the unique characteristics of adult stem cells isolated from hair follicles is their ability to differentiate into keratinocytes. Study on chitosan skin-engineered templates (SETs) as scaffolds for the proliferation of human fibroblasts have shown the promise of SETs in facilitating skin cell growth in three-dimensional culture. High proliferation in three-dimensional culture using human cells allows the researcher to extensively evaluate the cultivation of desirable cell types on chitosan SETs. Therefore, this study aimed to evaluate the in vitro attachment, proliferation and differentiation of hair follicle stem cells (HFSCs) on a chitosan SETs. HFSCs were isolated from human scalp tissues using collagenase type I prior to propagation in supplemented CnT-07 media. The phenotype of the HFSCs was verified using the markers keratin-15 (K15) and CD200, as detected by immunocytochemical staining. The attachment and proliferation of the HFSCs on the chitosan SETs were evaluated using scanning electron microscopy (SEM), an alamar blue assay and a live/dead assay. Subsequently, the HFSCs were differentiated using CnT-2D differentiation media. The cells’ differentiation was verified using the markers involucrin and keratin-6 (K6), as detected by immunofluorescence staining. The HFSCs were successfully isolated, proliferated and differentiated according to staining positivity and microscopy imaging. HFSCs are able to proliferate and directly differentiate into keratinocytes on a chitosan SETs, which may facilitate their use in regenerative medicine.

Isolation and In Vitro Characterization of Epidermal Stem Cells.

Colony-forming assays represent prospective methods, where cells isolated from enzymatically dissociated tissues or from tissue cultures are assessed for their proliferative capacity in vitro. Complex tissues such as the epithelial component of the skin (the epidermis) are characterized by a substantial cellular heterogeneity. Analysis of bulk populations of cells by colony-forming assays can consequently be convoluted by a number of factors that are not controlled for in population wide studies. It is therefore advantageous to refine in vitro growth assays by sub-fractionation of cells using flow cytometry. Using markers that define the spatial origin of epidermal cells, it is possible to interrogate the specific characteristics of subpopulations of cells based on their in vivo credentials. Here, we describe how to isolate, culture, and characterize keratinocytes from murine back and tail skin sorted by surface antigens associated with adult stem cell characteristics.

Abstract 2230: Wnt signaling in prostate cancer stem like cells

Abstract Introduction: Cancer stem like cells (CSLCs) are characterized by features of tumorigenicity, serial passages and the expression of specific stem cell markers. CSLCs possess characteristics of adult stem cells, including self-renewal. CSLCs can be isolated from cancer cell lines, including prostate, and form morphologically distinct colonies (e.g. holo, mero and para clones). Colony formation has been used as a surrogate biological assay to identify and characterize CSLCs and holo and meroclones from prostate cell lines have been proposed to contain CSLCs. Wnt signaling through the action of two major intracellular transducers, calcium ([Ca2+]i) and the transcription factor ß-catenin, is a key regulator of stem cell self-renewal. In whole populations of cancer cell lines (e.g. PC3, MCF7), Wnt signaling operates via a convergent rather than distinct canonical (Wnt/ß-catenin) and non-canonical (Wnt/Ca2+) pathways. Little is known about the characteristics of Wnt signaling (e.g. Wnt induced [Ca2+]i release or ß-catenin expression) in prostate CSLCs. The aim of this study was to characterize the clonal expansion of PC3 cell line and to investigate Wnt signaling in these colonies. Methods: PC3 cells, in a single cell suspension, were plated at low density (6 cells/cm2) in 6 well plates (Nunc) and imaged over 10 days with Incucyte (Essen Bioscience) and analysed using Image J software. Some colonies were loaded with Fluo4 and FuraRed dyes (Invitrogen) and used in a live [Ca2+]i imaging assay ± Wnt 5A or 9B (200ng/ml) with an Olympus FV1000 confocal system; the same colonies were then used in immunostaining for the ß-catenin protein (Abcam 22656) followed by confocal microscopy. Data is presented as means ± SE; Mann Whitney U test was used for statistical analysis. Results: IncuCyte imaging captured colony formation from single cells plated and two morphologically distinct types of PC3 colonies were observed at day 10, termed holoclone/meroclone (h/m clone) and paraclone (p clone). Mean colony diameter of those categorized as h/m clone was 838 ± 22μm (n = 102) and 1191 ± 28μm for p clone (n = 96); mean cell density (%) showed a significant difference (p<0.01) in h/m compared to p clones: 27.2 ± 3.2(n = 8) vs 4.7 ± 0.4 (n = 5), respectively. Cells in h/m and p clones showed [Ca2+]i release in response to both Wnt 5A and 9B; Cells in h/m clones demonstrated a significantly (p<0.05) longer dwell time for [Ca2+]i release in response to Wnt 9B than 5A, with mean dwell times (seconds) of 45.3 ± 2.6 (n = 52) and 38.0 ± 4.3 (n = 52), respectively.ß-catenin was expressed in both h/m and p clones from PC3 cell line. Conclusions: Distinct colony types can be observed in cultures of PC3 prostate cancer cell line. These colonies have quantifiable characteristics in addition of observable morphologies. A majority of cells within different colonies show Wnt induced [Ca2+]i release and ß-catenin expression. A convergent model of Wnt signaling is likely to operate in prostate CSLCs. Citation Format: Reena Davda, Christopher Thrasivoulou, John Masters, Aamir Ahmed. Wnt signaling in prostate cancer stem like cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2230. doi:10.1158/1538-7445.AM2015-2230

Mechanical stimulation of human tendon stem/progenitor cells results in upregulation of matrix proteins, integrins and MMPs, and activation of p38 and ERK1/2 kinases.

BackgroundTendons are dense connective tissues subjected periodically to mechanical stress upon which complex responsive mechanisms are activated. These mechanisms affect not only the development of these tissues but also their healing. Despite of the acknowledged importance of the mechanical stress for tendon function and repair, the mechanotransduction mechanisms in tendon cells are still unclear and the elucidation of these mechanisms is a key goal in tendon research. Tendon stem/progenitor cells (TSPC) possess common adult stem cell characteristics, and are suggested to actively participate in tendon development, tissue homeostasis as well as repair. This makes them an important cell population for tendon repair, and also an interesting research target for various open questions in tendon cell biology. Therefore, in our study we focused on TSPC, subjected them to five different mechanical protocols, and investigated the gene expression changes by using semi-quantitative, quantitative PCR and western blotting technologies.ResultsAmong the 25 different genes analyzed, we can convincingly report that the tendon-related genes - fibromodulin, lumican and versican, the collagen I-binding integrins - α1, α2 and α11, the matrix metalloproteinases - MMP9, 13 and 14 were strongly upregulated in TSPC after 3 days of mechanical stimulation with 8% amplitude. Molecular signaling analyses of five key integrin downstream kinases suggested that mechanical stimuli are mediated through ERK1/2 and p38, which were significantly activated in 8% biaxial-loaded TSPC.ConclusionsOur results demonstrate the positive effect of 8% mechanical loading on the gene expression of matrix proteins, integrins and matrix metalloproteinases, and activation of integrin downstream kinases p38 and ERK1/2 in TSPC. Taken together, our study contributes to better understanding of mechanotransduction mechanisms in TPSC, which in long term, after further translational research between tendon cell biology and orthopedics, can be beneficial to the management of tendon repair.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-015-0036-6) contains supplementary material, which is available to authorized users.

High throughput characterization of adult stem cells engineered for delivery of therapeutic factors for neuroprotective strategies.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system. Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system. Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Analysis of the stem cell characteristics of adult stem cells from Arbas white Cashmere goat

Studies have shown that multipotent adult stem cells possess differentiation characteristics similar to embryonic stem cells and pluripotent stem cells. We aimed to explore these similarities further by examining the expression of the pluripotency and stemness biomarkers, AKP, IL-6, Nanog, Oct-4, Rex-1, Sox-2 and TERT, as well as the triploblastic biomarkers, Sox-1, Myod1 and Gata-6 in adipose-derived stem cells (ADSCs), bone marrow stem cells (BMSCs) and muscle-derived satellite cells (MDSCs). These were isolated from adult Arbas white Cashmere goats and cultured in vitro. Immunocytochemistry, reverse transcription quantitative PCR and Western blotting were used to analyze the protein and mRNA expression of the markers. To investigate the ability of ADSCs, BMSCs and MDSCs to differentiate and cause tumors in vivo they were injected into immunodeficient mice (NOD-SCID). All results were compared to those for mouse embryonic stem cells (mESCs). Immunocytochemistry showed that AKP, IL-6, Nanog, Oct-4, Rex-1 and TERT were expressed in ADSCs, BMSCs and MDSCs, whereas Sox-2 was not. In ADSCs, the expression of IL-6 mRNA was relatively high, followed by Nanog and Oct-4, while Rex-1 and TERT expression were the lowest (P<0.01). In BMSCs, the expression of Rex-1 was relatively high, followed by IL-6, while Oct-4, Nanog and TERT were comparatively low (P<0.01). In MDSCs, the expression of IL-6, Nanog and Oct-4 were relatively high, while TERT was comparatively low (P<0.01). However, no expression of Sox-2 mRNA was detected in any of the three cell lines. The expression of Sox-1, Myod1 and Gata-6 was observed to different degrees in all three cell lines (P<0.01); the expression pattern in MDSCs was different from that in ADSCs and BMSCs. Western blotting indicated that no expression of Sox-2 and Rex-1 protein occurred in ADSCs, BMSCs and MDSCs, while the other five proteins were all expressed to different degrees (P<0.01); the expression pattern was consistent with the mRNA results. In contrast to the mESCs, no teratoma tissue or triploblastic differentiation appendages were formed in the immunodeficient mice after injection of ADSCs, BMSCs and MDSCs. Our results suggest that the three adult goat stem cell types are non-oncogenic and have stemness characteristics similar to embryonic stem cells. Of these, MDSCs were found to exhibit the most ESC-like properties and would make the best candidates for clinical application.

Isolation and Characterization of 2 New Human Rotator Cuff and Long Head of Biceps Tendon Cells Possessing Stem Cell–Like Self-Renewal and Multipotential Differentiation Capacity

Background: Stem cell therapy is expected to offer new alternatives to the traditional therapies of rotator cuff tendon tears. In particular, resident, tissue-specific, adult stem cells seem to have a higher regenerative potential for the tissue where they reside. Hypothesis: Rotator cuff tendon and long head of the biceps tendon possess a resident stem cell population that, when properly stimulated, may be induced to proliferate, thus being potentially usable for tendon regeneration. Study Design: Controlled laboratory study. Methods: Human tendon samples from the supraspinatus and the long head of the biceps were collected during rotator cuff tendon surgeries from 26 patients, washed with phosphate-buffered saline, cut into small pieces, and digested with collagenase type I and dispase. After centrifugation, cell pellets were resuspended in appropriate culture medium and plated. Adherent cells were cultured, phenotypically characterized, and then compared with human bone marrow stromal cells (BMSCs), as an example of adult stem cells, and human dermal fibroblasts, as normal proliferating cells with no stem cell properties. Results: Two new adult stem cell populations from the supraspinatus and long head of the biceps tendons were isolated, characterized, and cultured in vitro. Cells showed adult stem cell characteristics (ie, they were self-renewing in vitro, clonogenic, and multipotent), as they could be induced to differentiate into different cell types—namely, osteoblasts, adipocytes, and skeletal muscle cells. Conclusion: This work demonstrated that human rotator cuff tendon stem cells and human long head of the biceps tendon stem cells can be isolated and possess a high regenerative potential, which is comparable with that of BMSCs. Moreover, comparative analysis of the sphingolipid pattern of isolated cells with that of BMSCs and fibroblasts revealed the possibility of using this class of lipids as new possible markers of the cell differentiation status. Clinical Relevance: Rotator cuff and long head of the biceps tendons contain a stem cell population that can proliferate in vitro and could constitute an easily accessible stem cell source to develop novel therapies for tendon regeneration.

Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells

The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-27632, conditionally induces an indefinite proliferative state in primary mammalian epithelial cells. These conditionally reprogrammed cells (CRCs) are karyotype-stable and nontumorigenic. Because self-renewal is a recognized property of stem cells, we investigated whether Y-27632 and feeder cells induced a stem-like phenotype. We found that CRCs share characteristics of adult stem cells and exhibit up-regulated expression of α6 and β1 integrins, ΔNp63α, CD44, and telomerase reverse transcriptase, as well as decreased Notch signaling and an increased level of nuclear β-catenin. The induction of CRCs is rapid (occurs within 2 d) and results from reprogramming of the entire cell population rather than the selection of a minor subpopulation. CRCs do not overexpress the transcription factor sets characteristic of embryonic or induced pluripotent stem cells (e.g., Sox2, Oct4, Nanog, or Klf4). The induction of CRCs is also reversible, and removal of Y-27632 and feeders allows the cells to differentiate normally. Thus, when CRCs from ectocervical epithelium or tracheal epithelium are placed in an air-liquid interface culture system, the cervical cells form a well differentiated stratified squamous epithelium, whereas the tracheal cells form a ciliated airway epithelium. We discuss the diagnostic and therapeutic opportunities afforded by a method that can generate adult stem-like cells in vitro without genetic manipulation.

Abstract 3372: Mouse keratinocyte side-population plays an important role during malignant progression to skin squamous cell carcinomas

Abstract A subset of cells, termed side-population (SP), with characteristics of adult stem cells (SC) has been identified in several tissues, cell lines, as well as human and experimental tumors. The main feature of these putative stem cells is the high efflux capability for antimitotic drugs. The role of this SP in tumorigenesis is controversial; though, a role as cancer stem cells has been reported. Here, we have investigated whether a functionally equivalent SP exists in cell lines derived from mouse epidermis and skin tumors in order to define the extent of which the presence of SP is associated with tumor progression. We observed increased percentage of the SP in cell lines derived from skin squamous cell carcinomas (SCCs; cell lines CH72 and JWF2) compared with those arose from skin papillomas (308 and MT1/2) and mouse epidermis (C50). Side populations were isolated by the high capability to efflux Hoechst upon FACS analysis. Interestingly, qRT-PCR analysis of SP and non-SP shows elevated expression of the ABCG2/BCRP1 transporter -which participates in the dye efflux- in SP; while the expression of the hair follicle stem cell markers keratin 15 (K15), CD34 and Lgr6 were similar between SP and non-SP. These results suggest that the SP is not related to the well characterized stem cells from the bulge region of the hair follicle (BuSC). Importantly, the relevant correlation among the SP size and tumor progression suggests that these cells are positively selected during tumor progression. In order to localize the SP in mouse epidermis and tumors, we performed immunofluorescence analysis of skin papillomas and SCCs generated through the two-stage carcinogenesis protocol. We identified the ABCG2 transporter in a region near and above the BuSC area. Notably, this region was also positive for the thymic epithelial progenitor marker MTS24 which defines a keratinocyte population with characteristic of stem cells. Importantly, the ABCG2 transporter and MTS24 were localized in patches of cells on the proliferative basal cell layer of skin papillomas. Furthermore, we observed an elevated number of putative stem cells expressing ABCG2 and MTS24 in tumors from our previously published K5CDK4 mouse which showed increased malignant progression to SCC. Collectivelly, our results suggest that a putative stem cell population –different from the BuSCs–, play an important role during the malignant progression SCC. Supported by NIH grant CA116328. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3372. doi:1538-7445.AM2012-3372

Mechanical characterization of adult stem cells from bone marrow and perivascular niches

Therapies using adult stem cells often require mechanical manipulation such as injection or incorporation into scaffolds. However, force-induced rupture and mechanosensitivity of cells during manipulation is largely ignored. Here, we image cell mechanical structures and perform a biophysical characterization of three different types of human adult stem cells: bone marrow CD34+ hematopoietic, bone marrow mesenchymal and perivascular mesenchymal stem cells. We use micropipette aspiration to characterize cell mechanics and quantify deformation of subcellular structures under force and its contribution to global cell deformation. Our results suggest that CD34+ cells are mechanically suitable for injection systems since cells transition from solid- to fluid-like at constant aspiration pressure, probably due to a poorly developed actin cytoskeleton. Conversely, mesenchymal stem cells from the bone marrow and perivascular niches are more suitable for seeding into biomaterial scaffolds since they are mechanically robust and have developed cytoskeletal structures that may allow cellular stable attachment and motility through solid porous environments. Among these, perivascular stem cells cultured in 6% oxygen show a developed cytoskeleton but a more compliant nucleus, which can facilitate the penetration into pores of tissues or scaffolds. We confirm the relevance of our measurements using cell motility and migration assays and measure survival of injected cells. Since different types of adult stem cells can be used for similar applications, we suggest considering mechanical properties of stem cells to match optimal mechanical characteristics of therapies.

Systemic Delivery of Allogenic Muscle Stem Cells Induces Long-Term Muscle Repair and Clinical Efficacy in Duchenne Muscular Dystrophy Dogs

Duchenne muscular dystrophy (DMD) is a genetic progressive muscle disease resulting from the lack of dystrophin and without effective treatment. Adult stem cell populations have given new impetus to cell-based therapy of neuromuscular diseases. One of them, muscle-derived stem cells, isolated based on delayed adhesion properties, contributes to injured muscle repair. However, these data were collected in dystrophic mice that exhibit a relatively mild tissue phenotype and clinical features of DMD patients. Here, we characterized canine delayed adherent stem cells and investigated the efficacy of their systemic delivery in the clinically relevant DMD animal model to assess potential therapeutic application in humans. Delayed adherent stem cells, named MuStem cells (muscle stem cells), were isolated from healthy dog muscle using a preplating technique. In vitro, MuStem cells displayed a large expansion capacity, an ability to proliferate in suspension, and a multilineage differentiation potential. Phenotypically, they corresponded to early myogenic progenitors and uncommitted cells. When injected in immunosuppressed dystrophic dogs, they contributed to myofiber regeneration, satellite cell replenishment, and dystrophin expression. Importantly, their systemic delivery resulted in long-term dystrophin expression, muscle damage course limitation with an increased regeneration activity and an interstitial expansion restriction, and persisting stabilization of the dog's clinical status. These results demonstrate that MuStem cells could provide an attractive therapeutic avenue for DMD patients.

Mesenchymal stem cells and its Characterization

Although acceptable for cell based therapeutic applications, a rigorous understanding of the Mesenchymal Stem Cells (MSC) requires a better definition of what an MSC is. Inspite of the fact that no unique markers are known for MSCs, many attempts have been made to develop a cell-surface antigen profile for the better purification and identification of MSCs, particularly important is whether MSCs isolated from different tissues are identifiable by the same immunophenotype. While the precise identity of MSCs remains a challenge, further understanding of their biological properties will be greatly advanced by analyzing the mechanisms that govern their differentiation potential. The ability of MSCs to differentiate into a variety of connective tissue cell types has rendered them an ideal candidate cell source for clinical tissue regeneration strategies. This review focuses specifically in the context of these advances in characterization of adult stem cells via selection of unique cell surface markers and regulation of lineage- specific differentiation of mesenchymal stem cells.

Identification of single chain antibodies to breast cancer stem cells using phage display

Recent evidence suggests that most malignancies are driven by "cancer stem cells" sharing the signature characteristics of adult stem cells: the ability to self renew and to differentiate. Furthermore these cells are thought to be quiescent, infrequently dividing cells with a natural resistance to chemotherapeutic agents. These studies theorize that therapies, which effectively treat the majority of tumor cells but 'miss' the stem cell population, will fail, while therapies directed at stern cells can potentially eradicate tumors. In breast cancer, researchers have isolated 'breast cancer stem cells' capable of recreating the tumor in vivo and in vitro. Generated new tumors contained both additional numbers of cancer stem cells and diverse mixed populations of cells present in the initial tumor, supporting the intriguing self-renewal and differentiation characteristics. In the present study, an antibody phage library has been used to search for phage displayed-single chain antibodies (scFv) with selective affinity to specific targets on breast cancer stem cells. We demonstrate evidence of two clones binding specifically to a cancer stem cell population isolated from the SUMl59 breast cancer cell line. These clones had selective affinity for cancer stem cells and they were able to select cancer stem cells among a large population of non-stem cancer cells in paraffin-embedded sections. The applicability of these clones to paraffin sections and frozen tissue specimens made them good candidates to be used as diagnostic and prognostic markers in breast cancer patient samples taking into consideration the cancer stern cell concept in tumor biology.

Proliferation and Migration of Label-Retaining Cells of the Kidney Papilla

The kidney papilla contains a population of cells with several characteristics of adult stem cells, including the retention of proliferation markers during long chase periods (i.e., they are label-retaining cells [LRCs]). To determine whether the papillary LRCs generate new cells in the normal adult kidney, we examined cell proliferation throughout the kidney and found that the upper papilla is a site of enhanced cell cycling. Using genetically modified mice that conditionally expressed green fluorescence protein fused to histone 2B, we observed that the LRCs of the papilla proliferated only in its upper part, where they associate with "chains" of cycling cells. The papillary LRCs decreased in number with age, suggesting that the cells migrated to the upper papilla before entering the cell cycle. To test this directly, we marked papillary cells with vital dyes in vivo and found that some cells in the kidney papilla, including LRCs, migrated toward other parts of the kidney. Acute kidney injury enhanced both cell migration and proliferation. These results suggest that during normal homeostasis, LRCs of the kidney papilla (or their immediate progeny) migrate to the upper papilla and form a compartment of rapidly proliferating cells, which may play a role in repair after ischemic injury.

Self-Renewalof Stem Cells

Asymmetric division is one of the most fundamental characteristics of adult stem cells , which ensures one daughter cell maintains stem cell status and the other daughter cell becomes committed to differentiation. New data emerged recently that allow us to conclude that asymmetric division has another important aspect: it enables self-maintenance of stem cells.