Adult hair follicle stem cells do not retain the older DNA strands in vivo during normal tissue homeostasis

Tissue stem cells have been proposed to segregate the chromosomes asymmetrically (in a non-random manner), thereby retaining preferentially the older “immortal” DNA strands bearing the stemness characteristics into one daughter cell, whereas the newly synthesized strands are segregated to the other daughter cell that will commit to differentiation. Moreover, this non-random segregation would protect the stem cell genome from accumulating multiple mutations during repeated DNA replication. This long-standing hypothesis remains an active subject of study due to conflicting results for some systems and lack of consistency among different tissue stem cell populations. In this review, we will focus on work done in the hair follicle, which is one of the best-understood vertebrate tissue stem cell system to date. In cell culture analysis of paired cultured keratinocytes derived from hair follicle, stem cells suggested a non-random segregation of chromosome with respect to the older DNA strand. In vivo, the hair follicle stem cells appear to self-renew and differentiate at different phases of their homeostatic cycle. The fate decisions occur in quiescence when some stem cells migrate out of their niche and commit to differentiation without self-renewal. The stem cells left behind in the niche self-renew symmetrically and randomly segregate the chromosomes at each division, making more stem cells. This model seems to apply to at least a few other vertebrate tissue stem cells in vivo.     

Emerging targets for glioblastoma stem cell therapy

Glioblastoma multiforme (GBM), designated as World Health Organization (WHO) grade IV astrocytoma, is a lethal and therapy-resistant brain cancer comprised of several tumor cell subpopulations, including GBM stem cells (GSCs) which are believed to contribute to tumor recurrence following initial response to therapies. Emerging evidence demonstrates that GBM tumors are initiated from GSCs. The development and use of novel therapies including small molecule inhibitors of specific proteins in signaling pathways that regulate stemness, proliferation and migration of GSCs, immunotherapy, and non-coding microRNAs may provide better means of treating GBM. Identification and characterization of GSC-specific signaling pathways would be necessary to identify specific therapeutic targets which may lead to the development of more efficient therapies selectively targeting GSCs. Several signaling pathways including mTOR, AKT, maternal embryonic leucine zipper kinase (MELK), NOTCH1 and Wnt/b-catenin as well as expression of cancer stem cell markers CD133, CD44, Oct4, Sox2, Nanog, and ALDH1A1 maintain GSC properties. Moreover, the data published in the Cancer Genome Atlas (TCGA) specifically demonstrated the activated PI3K/AKT/mTOR pathway in GBM tumorigenesis. Studying such pathways may help to understand GSC biology and lead to the development of potential therapeutic interventions to render them more sensitive to chemotherapy and radiation therapy. Furthemore, recent demonstration of dedifferentiation of GBM cell lines into CSC-like cells prove that any successful therapeutic agent or combination of drugs for GBM therapy must eliminate not only GSCs, but the differentiated GBM cells and the entire bulk of tumor cells.     

c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation

The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. Here, we provide genetic evidence for an unexpected function of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors, suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term HSCs. Collectively, our data indicate that c-Myc controls the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSCs and their niche.     

Repopulating activity of ex vivo-expanded murine hematopoietic stem cells resides in the CD48-c-Kit+Sca-1+lineage marker- cell population

A better understanding of the biology of cultured hematopoietic stem cells (HSCs) is required to achieve ex vivo expansion of HSCs. In this study, clonal analysis of the surface phenotype and repopulating activity of ex vivo-expanded murine HSCs was performed. After 7 days of culture with stem cell factor, thrombopoietin, fibroblast growth factor-1, and insulin-like growth factor-2, single CD34-/lowc-Kit+Sca-1+lineage marker- (CD34-KSL) cells gave rise to various numbers of cells. The proportion of KSL cells decreased with increasing number of expanded cells. Transplantation studies revealed that the progeny containing a higher percentage of KSL cells tended to have enhanced repopulating potential. We also found that CD48 was heterogeneously expressed in the KSL cell population after culture. Repopulating activity resided only in the CD48-KSL cell population, which had a relatively long intermitotic interval. Microarray analysis showed surprisingly few differences in gene expression between cultured CD48-KSL cells (cycling HSCs) and CD48+KSL cells (cycling non-HSCs) compared with freshly isolated CD34-KSL cells (quiescent HSCs), suggesting that the maintenance of stem cell activity is controlled by a relatively small number of genes. These findings should lead to a better understanding of ex vivo-expanded HSCs.     

Deconstructing the hematopoietic stem cell niche: revealing the therapeutic potential

Development of the hematopoietic system is a stage-specific process where the bone marrow eventually becomes the principal source of hematopoiesis in the adult mammalian organism. Sustained hematopoiesis in the bone marrow, however, depends on the self-renewal of the resident hematopoietic stem cells (HSCs). The region where these HSCs are hypothesized to self renew is called the stem cell 'niche.' Recent studies have identified components of the HSC niche in the bone marrow, including cells of the osteoblastic lineage, extracellular matrix molecules and molecular signaling interactions between the stem cells and niche cells. Specific pharmacological targeting of these niche components has led to beneficial HSC effects, demonstrating a new therapeutic approach where stem cell function is altered through targeting of the niche.     

The niche as a target for hematopoietic manipulation and regeneration

Hematopoietic stem cells (HSCs), rare primitive cells capable of reconstituting all blood cell lineages, are the only stem cells currently routinely used for therapeutic purposes. Clinical experience has shown that HSC number is an important limiting factor in treatment success. Strategies to expand HSCs are of great clinical appeal, as they would improve therapeutic use of these cells in stem cell transplantation and in conditions of bone marrow failure. The microenvironment in which HSCs reside, known as the niche, has long been considered a critical regulator of HSCs. Data accumulated over the past decade strongly confirm the importance of the niche in HSC behavior. A number of niche components as well as signaling pathways, such as Notch, have been implicated in the interaction of the microenvironment with HSCs and continue to be genetically evaluated in the hope of defining the critical elements that are required and which, if modified, can initiate HSC behaviors. In this review, we highlight the known characteristics of HSCs, challenges in their expansion, the niche phenomenon, and explain why niche stimulated HSC expansion is of utmost interest in the field, while beginning to bring to the fore potential caveats of niche manipulation. Lastly, the potential pitfalls of avoiding malignancy and controlling self-renewal versus differentiation will be briefly reviewed.     

Two- and Three-Dimensional Culture of Keratinocyte Stem and Precursor Cells Derived from Primary Murine Epidermal Cultures

In the skin, multipotent keratinocyte stem cells (KSC) are localised in the hair follicle bulge region. Although, KSC can be cultivated and grown in two-dimensional (2D) culture they rapidly lose stem cell markers when isolated from their niche. Currently, there is no KSC culture method available which recapitulates an environment similar to the KSC niche in the hair follicle. Here we describe the successful establishment of an in vitro 3D stem cell culture model developed from clonally growing keratinocyte lines derived from neonatal mice using culture conditions previously established for human keratinocytes. After 20 passages, keratinocyte lines showed a stable ratio of holoclones (stem cells), meroclones (stem and precursor cells) and paraclones (differentiating cells), with approximately 29% holoclones, 54% meroclones and 17% paraclones, and were thus termed keratinocyte stem and precursor cell (KSPC) cultures. In high calcium medium, KSPC cultures grown at the air-liquid interphase differentiated and formed epidermal equivalents. Notably, and in contrast to primary keratinocytes, keratinocytes from KSPC cultures were able to aggregate and form spherical clusters in hanging drops, a characteristic hallmark shared with other stem cell types. Similar to the in vivo situation in the hair follicle bulge, KSPC aggregates also showed low proliferation, down-regulation of keratin 6, absence of keratin 1, and expression of the KSC markers keratin 15, Sox9, NFATc1 and Zfp145. KSPC aggregates therefore provide an optimal in vitro 3D environment for the further characterisation and study of normal and genetically modified KSPC.     

Enhanced self-renewal of hematopoietic stem cells mediated by the polycomb gene product Bmi-1

The Polycomb group (PcG) gene Bmi-1 has recently been implicated in the maintenance of hematopoietic stem cells (HSC) from loss-of-function analysis. Here, we demonstrate that increased expression of Bmi-1 promotes HSC self-renewal. Forced expression of Bmi-1 enhanced symmetrical cell division of HSCs and mediated a higher probability of inheritance of stemness through cell division. Correspondingly, forced expression of Bmi-1, but not the other PcG genes, led to a striking ex vivo expansion of multipotential progenitors and marked augmentation of HSC repopulating capacity in vivo. Loss-of-function analyses revealed that among PcG genes, absence of Bmi-1 is preferentially linked with a profound defect in HSC self-renewal. Our findings define Bmi-1 as a central player in HSC self-renewal and demonstrate that Bmi-1 is a target for therapeutic manipulation of HSCs.     

Overexpression of ZNF217 in glioblastoma contributes to the maintenance of glioma stem cells regulated by hypoxia-inducible factors

Glioblastoma multiforme (GBM) is the most aggressive and common kind of primary brain tumor in adults, and is thought to be driven by a subpopulation of glioma stem cells (GSCs). GSCs reside in a specialized hypoxic niche, which can regulate the tumorigenic capacity of GSCs primarily through the hypoxia-inducible factors (HIFs), HIF1α and HIF2α. ZNF217 is an oncogene frequently amplified in many kinds of tumors. It is associated with aggressive tumor behavior and poor clinical prognosis, but its role in gliomas is poorly known. Gene expression and copy number analysis from TCGA data reveal that ZNF217 is amplified in 32% and overexpressed in 71.2% of GBMs. Quantitative RT-PCR and western blotting of a cohort of glioma samples showed that ZNF217 was highly expressed in gliomas and increased with tumor grade. Analysis of a molecular database demonstrated that ZNF217 expression correlated with poor survival of glioma patients. Investigation of ZNF217 expression in GSCs, non-GSCs and normal neural stem cells (NSCs) indicated that ZNF217 was more highly expressed in GSCs than in non-GSCs and NSCs. Knockdown of ZNF217 in GSCs by small-interfering RNA (siRNA) inhibited their growth and promoted their differentiation. Interestingly, ZNF217 was upregulated in GSCs and the GBM cell line U87 when exposed to the hypoxic environment of 1% oxygen. Knockdown of either HIF1α or HIF2α, which has a central role in the hypoxia-induced responses of these cells, inhibited ZNF217 expression. In addition, ZNF217 upregulation was compromised under hypoxia in U87 and GSCs when either HIF1α or HIF2α was targeted by siRNA. HIF2α knockdown inhibited ZNF217 expression more efficiently in both normoxia and hypoxia than HIF1α knockdown. Therefore, ZNF217 is overexpressed in GBMs and contributes to the maintenance of GSCs, which is regulated by HIFs released by the hypoxic environment of the tumor.     

Neural Crest Stem Cell-specific Deletion of the Pygopus2 Gene Modulates Hair Follicle Development

We show that neural crest stem cells affect mouse hair follicle development. During embryogenesis hair follicle induction is regulated by complex reciprocal and functionally redundant signals between epidermis and dermis, which remain to be fully understood. Canonical Wnt signalling is a hallmark of neural crest cells and also a prerequisite for hair follicle induction prior to hair placode formation in the epidermis. As neural crest stem cells invade the epidermis during early embryonic development we aimed at determining whether neural crest cells affect hair follicle development. To attenuate, but not silence, canonical Wnt signalling specifically in neural crest cells, we analyzed Wnt1-cre(+/?)::Pygo2(?/?) mice in which the β-catenin co-activator gene, Pygopus 2 (Pygo2), is deleted specifically in neural crest cells. Both, hair density and hair thickness were reduced in mutant mice. Furthermore, hair development was delayed and the relative ratio of hair types was affected. There was a decrease in zig-zag hairs and an increase in awl hairs. Mouse neural crest stem cells expressed ectodysplasin, an essential effector in the formation of zig-zag hair. Taken together, our data support the novel notion that neural crest cells are involved in the earliest stages of hair follicle development.     

Biomimetic macroporous PEG hydrogels as 3D scaffolds for the multiplication of human hematopoietic stem and progenitor cells

Multiplication of hematopoietic stem cells (HSCs) in vitro with current standard methods is limited and mostly insufficient for clinical applications of these cells. They quickly lose their multipotency in culture because of the fast onset of differentiation. In contrast, HSCs efficiently self-renew in their natural microenvironment (their niche) in the bone marrow. Therefore, engineering artificial bone marrow analogs is a promising biomaterial-based approach for culturing these cells. In the current study, a straight-forward, easy-to-use method for the production of biofunctionalized, macroporous hydrogel scaffolds that mimic the spongy architecture of trabecular bones was developed. As surrogates for cellular components of the niche, mesenchymal stem cells (MSCs) from different sources (bone marrow and umbilical cord) and osteoblast-like cells were tested. MSCs from bone marrow had the strongest pro-proliferative effect on freshly isolated human hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood. Co-culture in the pores of the three-dimensional hydrogel scaffold showed that the positive effect of MSCs on preservation of HSPC stemness was more pronounced in 3D than in standard 2D cell culture systems. Thus, the presented biomimetic scaffolds revealed to meet the basic requirements for creating artificial HSC niches.     

The pathogenesis of primary cicatricial alopecias

Cicatricial (scarring) alopecia results from irreversible damage to epithelial stem cells located in the bulge region of the hair follicle, generally as a result of inflammatory mechanisms (eg, in the context of autoimmune disease). In primary cicactricial alopecia (PCA), the hair follicle itself is the key target of autoaggressive immunity. This group of permanent hair loss disorders can be classified into distinct subgroups, characterized by the predominant peri-follicular inflammatory cell type. In none of these PCA forms do we know exactly why hair follicles begin to attract such an infiltrate. Thus, it is not surprising that halting or even reversing this inflammation in PCA is often extremely difficult. However, increasing evidence suggests that healthy hair follicle epithelial stem cells enjoy relative protection from inflammatory assault by being located in an immunologically "privileged" niche. Because this protection may collapse in PCA, one key challenge in PCA research is to identify the specific signaling pathways that endanger, or restore, the relative immunoprotection of these stem cells. After a summary of pathobiological principles that underlie the development and clinical phenotype of PCA, we close by defining key open questions that need to be answered if more effective treatment modalities for this therapeutically very frustrating, but biologically fascinating, group of diseases are to be developed.     

Embryonic development of hair follicle pluripotent stem (hfPS) cells

Our laboratory discovered nestin-expressing hair follicle stem cells and demonstrated their pluripotency. We have shown that nestin-positive and K15-negative multipotent hair follicle stem cells are located above the hair follicle bulge, and we termed these cells hair follicle pluripotent stem (hfPS) cells. We have previously shown that hair follicle stem cells can regenerate peripheral nerve and spinal cord. In the present study, we describe the embryonic development of the hair follicle stem cell area (hfPSCA), which is located above the bulge and below the sebaceous glands in the adult mouse. At embryonic day 16.5 (E16.5) of nestindriven GFP (ND-GFP) transgenic mice, which express nestin in hfPS cells, the ND-GFP hair follicle stem cells are located in mesenchymal condensates. At postnatal day 0 (P0), the ND-GFP-expressing cells are migrating to the upper part of the hair follicle from the dermal papilla. At P3, keratin 15 (K15)-positive cells, derived from ND-GFP dermal papilla cells, are located in the outer-root sheath and basal layer of the epidermis. By P10, the ND-GFP have formed the K15-positive outer-root sheath as well as the ND-GFP hfPSA. These results suggest that ND-GFP hfPS cells in the dermal papilla form nestin-expressing hair follicle stem cells in the first hair cycle. These observations provide new insight into the origins of hfPS cells and the hfPSCA.     

Characterization of Rat Hair Follicle Stem Cells Selected by Vario Magnetic Activated Cell Sorting System

Hair follicle stem cells (HfSCs) play crucial roles in hair follicle morphogenesis and hair cycling. These stem cells are self-renewable and have the multi-lineage potential to generate epidermis, sebaceous glands, and hair follicle. The separation and identification of hair follicle stem cells are important for further research in stem cell biology. In this study, we report on the successful enrichment of rat hair follicle stem cells through vario magnetic activated cell sorting (Vario MACS) and the biological characteristics of the stem cells. We chose the HfSCs positive surface markers CD34, α6-integrin and the negative marker CD71 to design four isolation strategies: positive selection with single marker of CD34, positive selection with single marker of α6-integrin, CD71 depletion followed by CD34 positive selection, and CD71 depletion followed by α6-integrin positive selection. The results of flow cytometry analysis showed that all four strategies had ideal effects. Specifically, we conducted a series of researches on HfSCs characterized by their high level of CD34, termed CD34^bri cells, and low to undetectable expression of CD34, termed CD34^dim cells. CD34^bri cells had greater proliferative potential and higher colony-forming ability than CD34^dim cells. Furthermore, CD34^bri cells had some typical characteristics as progenitor cells, such as large nucleus, obvious nucleolus, large nuclear:cytoplasmic ratio and few cytoplasmic organelles. Our findings clearly demonstrated that HfSCs with high purity and viability could be successfully enriched with Vario MACS.     

Comparative characterization of hair follicle dermal stem cells and bone marrow mesenchymal stem cells

We compared the growth and differentiation characteristics of hair follicle-derived dermal stem cells with bone marrow mesenchymal stem cells (MSCs). Follicular dermal cells were isolated from whisker hairs of Wistar rats and bone marrow MSCs were isolated from femora of the same animals. The adherent hair follicle dermal cells showed a fibroblastic morphology in serum-containing culture medium, were CD44(+), CD73(+), CD90(+), and CD34(), and had a population doubling time of 27 h. MSCs isolated from the bone marrow showed a similar morphology and population doubling time and expressed the same cell-surface markers. Following exposure to appropriate induction stimuli, both cell populations had the capacity to differentiate into various mesenchymal lineages, such as osteoblasts, adipocytes, chondrocytes, and myocytes and expressed neuroprogenitor cell markers. The rate and extent of differentiation were remarkably similar for both hair follicleand bone marrow-derived cells, whereas interfollicular dermal cells failed to differentiate. We identified telomerase activity in follicle dermal stem cells and marrow MSCs and demonstrated that they were capable of clonal expansion. In ex vivo analyses, we identified the presence of putative dermal stem cells in the dermal sheath and dermal papillae of the hair follicle. Consequently, the hair follicle may represent a suitable, accessible source for MSCs.