Identification of new human mesenchymal stem cell biomarker by aptamers / Identificação de novos biomarcadores de células tronco mesenquimais de origem humana por meio de aptâmeros

Stem cells are undifferentiated cells that can be distinguished from others by their ability to self-renew and to differentiate into new specific cell types. Mesenchymal stem cells (MSC) are adult stem cells that can be obtained from different sources, such as adipose tissue, bone marrow, dental pulp, and umbilical cord. They can either replicate, originating new identical cells, or differentiate into cells of mesodermal origin and from other germ layers. MSC have been studied as new tools for regenerative therapy. Although encouraging results have been demonstrated, MSC-based therapies still face a great barrier: the difficulty of isolating these cells from heterogeneous environments. MSC are currently characterized by immunolabelling through a set of multiple surface membrane markers, including CD29, CD73, CD90 and CD105, which are also expressed by other cell types. Hence, the present work aimed to identify new specific biomarkers for the characterization of human MSC using DNA aptamers produced by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique. Our results showed that MSC from different origins bound to DNA candidate aptamers, that is, DNA or RNA oligonucleotides selected from random libraries that bind specifically to biological targets. Aptamer-bound MSC could be isolated by fluorescenceactivated cell sorting (FACS) procedures, enhancing the induction of differentiation into specific phenotypes (chondrocytes, osteocytes and adipocytes) when compared to the whole MSC population. Flow cytometry analyses revealed that candidate aptamers bound to 50% of the MSC population from dental pulp and did not present significant binding rates to human fibroblasts or lymphocytes, both used as negative control. Moreover, immunofluorescence images and confocal analyses revealed staining of MSC by aptamers localized in the surfacemembrane of these cells. The results also showed internal staining of human monocytes by our investigated aptamers. A non-specific control aptamer (CNTR APT) obtained from the random pool was then utilized to compare the specificity of the aptamers bound to the analyzed non-apoptotic cells, showing no staining for MSC. However, 40% of the monocytes bound to the CNTR APT. Normalized data based on the cells bound to candidate aptamers compared to those bound to the CNTR APT, revealed a 10 to 16-fold higher binding rate for MSC against 2-fold for monocytes. Despite its low specificity, monocyte-aptamer binding occurs probably due to the expression of shared markers with MSC, since monocytes are derived from hematopoietic stem cells and are important for the immune system ability to internalize/phagocyte external molecules. Given that, we performed a pull-down assay followed by mass spectrometry analysis to detect which MSC-specific protein or other target epitope not coexpressed by monocytes or the CNTR APT would bind to the candidate aptamer. Distinguishing between MSC and monocyte epitopes is important, as both cells are involved in immunomodulatory effects after MSC transplantations. ADAM17 was found to be a target of the APT10, emerging as a possible biomarker of MSC, since its involvement in the inhibition of the TGF signaling cascade, which is responsible for the differentiation of MSC. Thus, MSC with a higher stemness profile should overexpress the protein ADAM17, which presents a catalytic site with affinity to APT10. Another target of Apt 10 is VAMP3, belonging to a transmembrane protein complex that is involved in endocytosis and exocytosis processes during immune and inflammatory responses. Overall, proteins identified as targets of APT10 may be cell surface MSC biomarkers, with importance for MSC-based cell and immune therapies

Células tronco são células indiferenciadas que podem ser distinguidas de outros tipos celulares por meio da habilidade de se auto renovarem e de se diferenciarem em novos tipos celulares. Células tronco mesenquimais (MSC) são células tronco adultas encontradas em diferentes tecidos como tecido adiposo, polpa de dente e cordão umbilical. Estas células podem se autodividir em células idênticas ou se diferenciarem em células de origem mesodermal. Estas células têm sido estudadas em novas aplicações que envolvem terapia regenerativas. Embora resultados encorajadores tenham sido demonstrados, terapias que utilizam MSC ainda encontram uma grande barreira: a dificuldade no isolamento destas células a partir de um ambiente heterogêneo. MSC são caracterizadas por populações positivas em ensaios de imunomarcação para os epítopos membranares CD29, CD73, CD90 e CD105, presentes também em outros tipos celulares. Assim, o presente trabalho tem o objetivo de identificar novos biomarcadores de MSC de origem humana, utilizando aptâmeros de DNA produzidos pela técnica SELEX (Systematic Evolution of Ligands by EXponential Enrichment) como ferramenta. Nossos resultados mostraram que MSC de diferentes origens ligam-se a aptâmeros (oligonucleotídeos de DNA ou RNA que atuam como ligantes específicos de alvos moleculares) de DNA candidatos que atuam no isolamento de MSC por meio da técnica FACS de separação celular, promovendo uma maior indução de diferenciação em células específicas (condrócitos, osteócitos e adipócitos) comparada com a população total de MSC. Análises de citometria de fluxo mostraram que os aptâmeros candidatos se ligam a 50% das MSC de polpa de dente e não apresentam taxa de ligação significante para fibroblastos e linfócitos de origem humana - utilizados como controles negativo. Além domais, imagens de imunofluorescência e confocal mostraram ligação na superfície da membrana de MSC e a marcação interna de monócitos a estes aptâmeros. Portanto, um aptâmero controle (CNTR APT) foi utilizado para comparar a especificidade dos aptâmeros ligados a células viáveis, mostrando a não ligação deste aptâmero a MSC. Porém, 40% da população de monócitos ligou-se ao CNTR APT. Uma normalização baseada na comparação entre as taxas de ligação entre células ligadas com aptâmeros candidatos e o aptâmero controle gerou uma taxa de especificidade entre 10-16 vezes maior para MSC contra 2,5 vezes para os monócitos. Deste modo, embora os resultados tenham mostrado uma taxa de ligação entre monócitos e aptâmeros, as MSC ligadas aos aptâmeros candidatos possuem uma maior taxa de especificidade devido a uma maior presença de antígenos que são expressos em ambas as células. Um ensaio de Pull Down seguido de espectrometria de massas foi utilizado para a identificação de biomarcadores que se ligariam aos aptâmeros candidatos, e que não seriam co-expressos por monócitos e por antígenos ligados ao aptâmero controle. Deste modo, a proteína ADAM17 foi identificada nas amostras de APT10 ligadas às MSC. Tal proteína está relacionada à inibição de uma cascata de sinalização da família de proteínas TGF, responsável pela diferenciação de MSC. Assim, MSC com maior potencial tronco deveriam expressar ADAM17 em maior quantidade. Tal proteína apresenta um sítio catalítico que demonstra interagir com o APT10, de acordo com predição Docking entre proteína e DNA. Foi identificada também, a proteína VAMP3, que pertence a um complexo proteico transmembranar responsável pelos processos de endocitose e exocitose, e que podem ter um papel importante na liberação de citocinas e outras moléculas relacionadas às respostas imune e inflamatórias. Deste modo, o APT10 identificou proteínas importantes que devem estar relacionas com a melhora de imunoterapias que utilizam MSC

Whole transcriptome analysis of bovine mammary progenitor cells by P-Cadherin enrichment as a marker in the mammary cell hierarchy.

Adult bovine mammary stem cells possess the ability to regenerate in vivo clonal outgrowths that mimic functional alveoli. Commonly available techniques that involve immunophenotype-based cell sorting yield cell fractions that are moderately enriched, far from being highly purified. Primary bovine mammary epithelial cells segregated in four different populations according to the expression of P-Cadherin and CD49f. Sorted cells from each fraction were tested for the presence of lineage-restricted progenitors and stem cells. Only cells from the CD49fhigh/P-Cadherinneg subpopulation were able to give rise to both luminal- and myoepithelial-restricted colonies in vitro and generate organized outgrowths in vivo, which are hallmarks of stem cell activity. After whole transcriptome analysis, we found gene clusters to be differentially enriched that relate to cell-to-cell communication, metabolic processes, proliferation, migration and morphogenesis. When we analyzed only the genes that were differentially expressed in the stem cell enriched fraction, clusters of downregulated genes were related to proliferation, while among the upregulated expression, cluster of genes related to cell adhesion, migration and cytoskeleton organization were observed. Our results show that P-Cadherin separates mammary subpopulations differentially in progenitor cells or mammary stem cells. Further we provide a comprehensive observation of the gene expression differences among these cell populations which reinforces the assumption that bovine mammary stem cells are typically quiescent.

A High-Resolution Proteomic Landscaping of Primary Human Dental Stem Cells: Identification of SHED- and PDLSC-Specific Biomarkers.

Dental stem cells (DSCs) have emerged as a promising tool for basic research and clinical practice. A variety of adult stem cell (ASC) populations can be isolated from different areas within the dental tissue, which, due to their cellular and molecular characteristics, could give rise to different outcomes when used in potential applications. In this study, we performed a high-throughput molecular comparison of two primary human adult dental stem cell (hADSC) sub-populations: Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) and Periodontal Ligament Stem Cells (PDLSCs). A detailed proteomic mapping of SHEDs and PDLSCs, via employment of nano-LC tandem-mass spectrometry (MS/MS) revealed 2032 identified proteins in SHEDs and 3235 in PDLSCs. In total, 1516 proteins were expressed in both populations, while 517 were unique for SHEDs and 1721 were exclusively expressed in PDLSCs. Further analysis of the recorded proteins suggested that SHEDs predominantly expressed molecules that are involved in organizing the cytoskeletal network, cellular migration and adhesion, whereas PDLSCs are highly energy-producing cells, vastly expressing proteins that are implicated in various aspects of cell metabolism and proliferation. Applying the Rho-GDI signaling pathway as a paradigm, we propose potential biomarkers for SHEDs and for PDLSCs, reflecting their unique features, properties and engaged molecular pathways.

Comparing adult renal stem cell identification, characterization and applications.

Despite growing interest and effort, a consensus has yet to be reached in regards to the identification of adult renal stem cells. Organ complexity and low turnover of renal cells has made stem cell identification difficult and lead to the investigation of multiple possible populations. In this review, we summarize the work that has been done toward finding and characterizing an adult renal stem cell population. In addition to giving a general overview of what has been done, we aim to highlight the variation in methods and outcomes. The methods used to locate potential stem cell populations can vary widely, but even within the relatively standard practice of BrdU labeling of slowly dividing cells, there are significant differences in protocols and results. Additional diversity exists in cell marker profiles and apparent differentiation potential seen in potential stem cell sources. Cataloging the variety of methods and outcomes seen so far may help to streamline future investigation and stear the field toward consensus. But even without firmly defined populations, the application of renal stem cells holds tantalizing potential. Populations of highly proliferative, multipotent cells of renal origin show the ability to engraft in injured kidneys, mitigate functional loss and occasionally show the ability to generate nephrons de novo. The progress toward regenerative medicine applications is also summarized.

Skin Stem Cells: At the Frontier Between the Laboratory and Clinical Practice. Part 1: Epidermal Stem Cells.

Stem cells are characterized by their ability to self-renew and differentiate into the different cell lineages of their tissue of origin. The discovery of stem cells in adult tissues, together with the description of specific markers for their isolation, has opened up new lines of investigation, expanding the horizons of biomedical research and raising new hope in the treatment of many diseases. In this article, we review in detail the main characteristics of the stem cells that produce the specialized cells of the skin (epidermal, mesenchymal, and melanocyte stem cells) and their potential implications and applications in diseases affecting the skin. Part I deals with the principal characteristics and potential applications of epidermal stem cells in dermatology.

Embryonic Origin of Postnatal Neural Stem Cells.

Adult neural stem/progenitor (B1) cells within the walls of the lateral ventricles generate different types of neurons for the olfactory bulb (OB). The location of B1 cells determines the types of OB neurons they generate. Here we show that the majority of mouse B1 cell precursors are produced between embryonic days (E) 13.5 and 15.5 and remain largely quiescent until they become reactivated postnatally. Using a retroviral library carrying over 100,000 genetic tags, we found that B1 cells share a common progenitor with embryonic cells of the cortex, striatum, and septum, but this lineage relationship is lost before E15.5. The regional specification of B1 cells is evident as early as E11.5 and is spatially linked to the production of neurons that populate different areas of the forebrain. This study reveals an early embryonic regional specification of postnatal neural stem cells and the lineage relationship between them and embryonic progenitor cells.

IGF1 stimulates crypt expansion via differential activation of 2 intestinal stem cell populations.

Insulin-like growth factor 1 (IGF1) has potent trophic effects on normal or injured intestinal epithelium, but specific effects on intestinal stem cells (ISCs) are undefined. We used Sox9-enhanced green fluorescent protein (EGFP) reporter mice that permit analyses of both actively cycling ISCs (Sox9-EGFP(Low)) and reserve/facultative ISCs (Sox9-EGFP(High)) to study IGF1 action on ISCs in normal intestine or during crypt regeneration after high-dose radiation-induced injury. We hypothesized that IGF1 differentially regulates proliferation and gene expression in actively cycling and reserve/facultative ISCs. IGF1 was delivered for 5 days using subcutaneously implanted mini-pumps in uninjured mice or after 14 Gy abdominal radiation. ISC numbers, proliferation, and transcriptome were assessed. IGF1 increased epithelial growth in nonirradiated mice and enhanced crypt regeneration after radiation. In uninjured and regenerating intestines, IGF1 increased total numbers of Sox9-EGFP(Low) ISCs and percentage of these cells in M-phase. IGF1 increased percentages of Sox9-EGFP(High) ISCs in S-phase but did not expand this population. Microarray revealed that IGF1 activated distinct gene expression signatures in the 2 Sox9-EGFP ISC populations. In vitro IGF1 enhanced enteroid formation by Sox9-EGFP(High) facultative ISCs but not Sox9-EGFP(Low) actively cycling ISCs. Our data provide new evidence that IGF1 activates 2 ISC populations via distinct regulatory pathways to promote growth of normal intestinal epithelium and crypt regeneration after irradiation.

Unraveling intestinal stem cell behavior with models of crypt dynamics.

The definition, regulation and function of intestinal stem cells (ISCs) has been hotly debated. Recent discoveries have started to clarify the nature of ISCs, but many questions remain. This review discusses the current advances and controversies of ISC biology as well as theoretical compartmental models that have been coupled with in vivo experimentation to investigate the mechanisms of ISC dynamics during homeostasis, tumorigenesis, repair and development. We conclude our review by discussing the key lingering questions in the field and proposing how many of these questions can be addressed using both compartmental models and experimental techniques.

Aß increases neural stem cell activity in senescence-accelerated SAMP8 mice.

Neurogenesis persists in the adult brain as a form of plasticity due to the existence of neural stem cells (NSCs). Alterations in neurogenesis have been found in transgenic Alzheimer's disease (AD) mouse models, but NSC activity and neurogenesis in sporadic AD models remains to be examined. We herein describe a remarkable increase in NSC proliferation in the forebrain of SAMP8, a non-transgenic mouse strain that recapitulates the transition from healthy aging to AD. The increase in proliferation is transient, precedes AD-like symptoms such as amyloid beta 1-42 [Aß(1-42)] increase or gliosis, and is followed by a steep decline at later stages. Interestingly, in vitro studies indicate that secreted Aß(1-42) and PI3K signaling may account for the early boost in NSC proliferation. Our results highlight the role of soluble Aß(1-42) peptide and PI3K in the autocrine regulation of NSCs, and further suggest that over-proliferation of NSCs before the appearance of AD pathology may underlie neurogenic failure during the age-related progression of the disease. These findings have implications for therapeutic approaches based on neurogenesis in AD.

Differences in gene expression and cytokine release profiles highlight the heterogeneity of distinct subsets of adipose tissue-derived stem cells in the subcutaneous and visceral adipose tissue in humans.

Differences in the inherent properties of adipose tissue-derived stem cells (ASC) may contribute to the biological specificity of the subcutaneous (Sc) and visceral (V) adipose tissue depots. In this study, three distinct subpopulations of ASC, i.e. ASCSVF, ASCBottom, and ASCCeiling, were isolated from Sc and V fat biopsies of non-obese subjects, and their gene expression and functional characteristics were investigated. Genome-wide mRNA expression profiles of ASCSVF, ASCBottom and ASCCeiling from Sc fat were significantly different as compared to their homologous subsets of V-ASCs. Furthermore, ASCSVF, ASCCeiling and ASCBottom from the same fat depot were also distinct from each other. In this respect, both principal component analysis and hierarchical clusters analysis showed that ASCCeiling and ASCSVF shared a similar pattern of closely related genes, which was highly different when compared to that of ASCBottom. However, larger variations in gene expression were found in inter-depot than in intra-depot comparisons. The analysis of connectivity of genes differently expressed in each ASC subset demonstrated that, although there was some overlap, there was also a clear distinction between each Sc-ASC and their corresponding V-ASC subsets, and among ASCSVF, ASCBottom, and ASCCeiling of Sc or V fat depots in regard to networks associated with regulation of cell cycle, cell organization and development, inflammation and metabolic responses. Finally, the release of several cytokines and growth factors in the ASC cultured medium also showed both inter- and intra-depot differences. Thus, ASCCeiling and ASCBottom can be identified as two genetically and functionally heterogeneous ASC populations in addition to the ASCSVF, with ASCBottom showing the highest degree of unmatched gene expression. On the other hand, inter-depot seem to prevail over intra-depot differences in the ASC gene expression assets and network functions, contributing to the high degree of specificity of Sc and V adipose tissue in humans.

Functional cell types in taste buds have distinct longevities.

Taste buds are clusters of polarized sensory cells embedded in stratified oral epithelium. In adult mammals, taste buds turn over continuously and are replenished through the birth of new cells in the basal layer of the surrounding non-sensory epithelium. The half-life of cells in mammalian taste buds has been estimated as 8-12 days on average. Yet, earlier studies did not address whether the now well-defined functional taste bud cell types all exhibit the same lifetime. We employed a recently developed thymidine analog, 5-ethynil-2'-deoxyuridine (EdU) to re-evaluate the incorporation of newly born cells into circumvallate taste buds of adult mice. By combining EdU-labeling with immunostaining for selected markers, we tracked the differentiation and lifespan of the constituent cell types of taste buds. EdU was primarily incorporated into basal extragemmal cells, the principal source for replenishing taste bud cells. Undifferentiated EdU-labeled cells began migrating into circumvallate taste buds within 1 day of their birth. Type II (Receptor) taste cells began to differentiate from EdU-labeled precursors beginning 2 days after birth and then were eliminated with a half-life of 8 days. Type III (Presynaptic) taste cells began differentiating after a delay of 3 days after EdU-labeling, and they survived much longer, with a half-life of 22 days. We also scored taste bud cells that belong to neither Type II nor Type III, a heterogeneous group that includes mostly Type I cells, and also undifferentiated or immature cells. A non-linear decay fit described these cells as two sub-populations with half-lives of 8 and 24 days respectively. Our data suggest that many post-mitotic cells may remain quiescent within taste buds before differentiating into mature taste cells. A small number of slow-cycling cells may also exist within the perimeter of the taste bud. Based on their incidence, we hypothesize that these may be progenitors for Type III cells.

Association of HIV-Infection and antiretroviral therapy with levels of endothelial progenitor cells and subclinical atherosclerosis.

BACKGROUND: Although in the general population circulating vascular progenitor cell levels have been implicated in the homeostasis of the vascular wall through differentiation into endothelium and/or smooth muscle cells, it has not yet been assessed in HIV-infected patients. We herein investigated the number of progenitor cell subpopulations in HIV-infected patients and its relationship to carotid intima-media thickness (c-IMT). METHODS: Blood samples were collected from 200 HIV-infected patients and CD34/KDR, CD34/VE-cadherin, and CD14/Endoglin progenitor cells were identified by flow cytometry. c-IMT was determined by ultrasonography. A group of 27 healthy subjects was used as control group. RESULTS: In our population (20 ART-naive patients and 180 treated patients), traditional cardiovascular risk factors were not found predictive of vascular progenitor cell levels. However, antiretroviral therapy (ART)-treatment was identified as the main predictive value for low CD34/KDR cells and high CD14/Endoglin cells after adjustment by cardiovascular risk factors (age, sex, hypertension, diabetes, and hyperlipidaemia) and HIV-related characteristics (HIV duration and ART treatment). Low levels of circulating CD34/KDR or CD34/VE-cadherin endothelial progenitor cells tended to be associated with increased c-IMT. However, a positive association was found between CD14/Endoglin cells and c-IMT. Low number of CD34/KDR cells was also associated with the longest exposure to nucleoside reverse transcriptase inhibitors and/or protease inhibitors. CONCLUSIONS: ART exposure is the main predictor of circulating vascular progenitor cell levels. However, their levels are only partially associated with high c-IMT in HIV-infected patients. ART has already been found to have proatherogenic effect, but our data first describe its relationship with vascular progenitor cells and c-IMT.

Identification of stem cell populations in sweat glands and ducts reveals roles in homeostasis and wound repair.

Sweat glands are abundant in the body and essential for thermoregulation. Like mammary glands, they originate from epidermal progenitors. However, they display few signs of cellular turnover, and whether they have stem cells and tissue-regenerative capacity remains largely unexplored. Using lineage tracing, we here identify in sweat ducts multipotent progenitors that transition to unipotency after developing the sweat gland. In characterizing four adult stem cell populations of glandular skin, we show that they display distinct regenerative capabilities and remain unipotent when healing epidermal, myoepithelial-specific, and lumenal-specific injuries. We devise purification schemes and isolate and transcriptionally profile progenitors. Exploiting molecular differences between sweat and mammary glands, we show that only some progenitors regain multipotency to produce de novo ductal and glandular structures, but that these can retain their identity even within certain foreign microenvironments. Our findings provide insight into glandular stem cells and a framework for the further study of sweat gland biology.

Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.

As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy.

Stem cell therapy: a challenge to periodontist.

Periodontitis is an inflammatory disease which manifests clinically as loss of supporting periodontal tissues including periodontal ligament, cementum, and alveolar bone, and periodontal therapy is aimed at achieving complete regeneration of these structures. To date, this goal has been tried to accomplish using various bone grafts, growth factors, and barrier membranes. Stem cells are the most fascinating area of biology today and have been used clinically in the field of medicine to treat many incurable diseases. Various human and animal studies have confirmed the presence of stem cells in dental tissues including periodontal ligament. This has opened new avenues aiming toward complete periodontal regeneration using cell-based therapies. This review provides an overview of various types of stem cells in medicine and dentistry and their potential uses especially pertaining to periodontal regeneration.

Stem cells isolated from human dental follicles have osteogenic potential.

OBJECTIVE: Stem cells isolated from human dental follicles as a potential cell source for bone-tissue engineering were examined for correcting a critical bone defect. STUDY DESIGN: Impacted third molars were collected and single cell-derived cell populations were cultivated in growth medium. Single cell-derived cell lines were examined in terms of cell shape, gene expression patterns, differentiation capacity in vitro, and osteogenic potential in vivo. RESULTS: Three distinct cell populations were identified with different morphologies, patterns of gene expression, and differentiation capacity. All 3 cell populations promoted bone formation when transplanted into surgically created critical-size defects in immunodeficient rat calvaria, compared with control animals without cell transplantation, although one of these populations showed a weak capacity for osteogenetic differentiation in vitro. CONCLUSIONS: Human dental follicle can derive at least 3 unique cell populations in culture, all of which promote bone formation in vivo.

Inherent differential propensity of dental pulp stem cells derived from human deciduous and permanent teeth.

INTRODUCTION: Lately, several new stem cell sources and their effective isolation have been reported that claim to have potential for therapeutic applications. However, it is not yet clear which type of stem cell sources are most potent and best for targeted therapy. Lack of understanding of nature of these cells and their lineage-specific propensity might hinder their full potential. Therefore, understanding the gene expression profile that indicates their lineage-specific proclivity is fundamental to the development of successful cell-based therapies. METHODS: We compared proliferation rate, gene expression profile, and lineage-specific propensity of stem cells derived from human deciduous (SCD) and permanent teeth (DPSCs) over 5 passages. RESULTS: The proliferation rate of SCD was higher (cell number, 25 x 10(6) cells/mL; percent colony-forming units [CFUs], 151.67 +/- 10.5; percent cells in S/G2 phase, 12.4 +/- 1.48) than that of DPSCs (cell number, 21 x 10(6) cells/mL; percent CFUs, 133 +/- 17.62; percent cells in S/G2 phase, 10.4 +/- 1.18). It was observed that fold expression of several pluripotent markers such as OCT4, SOX2, NANOG, and REX1 were higher (>2) in SCD as compared with DPSCs. However, DPSCs showed higher expression of neuroectodermal markers PAX6, GBX2, and nestin (fold expression >100). Similarly, higher neurosphere formation and neuronal marker expression (NF, GFAP) were found in the differentiated DPSCs into neuron-like cells as compared with SCD. CONCLUSIONS: This study thus demonstrates that both SCD and DPSCs exhibit specific gene expression profile, with clear-cut inclination of DPSCs toward neuronal lineage.

A novel classification of quiescent and transit amplifying adult neural stem cells by surface and metabolic markers permits a defined simultaneous isolation.

Adult neural stem and progenitor cells (NSPCs) are usually defined retrospectively by their ability to proliferate in vivo (bromodeoxyuridine uptake) or to form neurospheres and to differentiate into neurons, astrocytes and oligodendrocytes in vitro. Additional strategies to identify and to isolate NSPCs are of great importance for the investigation of cell differentiation and fate specification. Using the cell surface molecules Prominin-1 and Lewis X and a metabolic marker, the aldehyde dehydrogenase activity, we isolated and characterized five main populations of NSPCs in the neurogenic subventricular zone (SVZ) and the non-neurogenic spinal cord (SC). We used clonal analysis to assess neurosphere formation and multipotency, BrdU retention to investigate in vivo proliferation activity and quantified the expression of NSPC associated genes. Surprisingly, we found many similarities in NSPC subpopulations derived from the SVZ and SC suggesting that subtypes with similar intrinsic potential exist in both regions. The marker defined classification of NSPCs will help to distinguish subpopulations of NSPCs and allows their prospective isolation using fluorescence activated cell sorting.