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Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

Newcastle University

Stem cells are clonogenic cells with self-renewal and differentiation properties, which may represent a major target for genetic damage leading to prostate cancer and benign prostatic hyperplasia. Stem cells remain poorly characterised because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying cells, which have a more restricted proliferative potential. Human CD133 antigen, also known as AC133, was recently identified as a haematopoietic stem cell marker. Here we show that a small population (approximately 1%) of human prostate basal cells express the cell surface marker CD133 and are restricted to the alpha(2)beta(1)(hi) population, previously shown to be a marker of stem cells in prostate epithelia. alpha(2)beta(1)(hi)/CD133(+) cells exhibit two important attributes of epithelial stem cells: they possess a high in vitro proliferative potential and can reconstitute prostatic-like acini in immunocompromised male nude mice.

CD133, a novel marker for human prostatic epithelial stem cells.

Cultured cells from the adult human hair follicle dermis can be directed toward adipogenic and osteogenic differentiation.

The dermal components of the hair follicle exhibit a number of stem cell properties, including regenerative potential, roles in wound healing and the ability to produce a functional dermis. Here we examine the stem cell phenomenon of plasticity, focusing on recent observations of in vitro plasticity of dermal papilla and sheath cells, including previously unpublished data of neuronal-like differentiation. We then briefly address the implications of the stem cell potential of hair follicle dermal cells for the field of tissue engineering.

Plasticity of rodent and human hair follicle dermal cells: implications for cell therapy and tissue engineering.

The mammalian epidermis is the first line of defense against external environmental challenges including dehydration. The epidermis undergoes a highly intricate developmental program in utero, transforming from a simple to a complex stratified epithelium. During this process of stratification and differentiation, epidermal keratinocytes express a defined set of structural proteins, mainly keratins, whose expression is controlled by largely unknown mechanisms. In order to identify novel factors contributing to epidermal morphogenesis, we performed a global transcriptional analysis of the developing mouse epidermis after separating it from the underlying dermis (E12.5-E15.5). Unexpectedly, the recently identified genes encoding secreted peptides dermokine (Dmkn), keratinocyte differentiation-associated protein (krtdap), and suprabasin (Sbsn) as well as a largely uncharacterized embryonic keratin (Krt77), were among the most highly differentially expressed genes. The three genes encoding the secreted proteins form a cluster in an approximately 40-Kb locus on human chromosome 19 and the syntenic region on mouse chromosome 7 known as the stratified epithelium secreted peptides complex (SSC). Using whole mount in situ hybridization, we show that these genes show a coordinated spatio-temporal expression pattern during epidermal morphogenesis. The expression of these genes initiates in the nasal epithelium and correlates with the initiation of other epidermal differentiation markers such as K1 and loricrin (Byrne et al. [1994] Development 120:2369-2383), as well as the initiation of barrier formation. Our observations reveal a coordinated mode of expression of the SSC genes as well as the correlation of their initiation in the nasal epithelium with the initiation of barrier formation at this site.

Transcriptional profiling of developing mouse epidermis reveals novel patterns of coordinated gene expression.

Ectodermal appendage morphogenesis requires continuous epithelial-mesenchymal cross-talk during development. Canonical Wnt signaling has been shown to be pivotal during this process and its inhibition leads to the absence of any morphological or molecular signs of appendage formation, including hair follicles (HFs). In the mouse, primary HFs arise in utero starting just before E14.5, when the first morphological signs of a placode are discernible. In this study, our goal was to identify novel factors expressed during primary HF morphogenesis. We performed transcriptional profiling of the developing epidermis at 12 h intervals between E12.5 and E15.5. One of the significantly differentially expressed genes was the Wnt inhibitor Dickkopf 4, Dkk4. We show that Dkk4 mRNA increases sharply in the dorso-lateral epidermis around E14 and then decreases until E15.5. Using whole mount in situ hybridization, we show that Dkk4 mRNA is localized to the pre-placodes at sites of presumptive epithelial-mesenchymal interactions during appendage morphogenesis, including the dental lamina, mammary gland, eccrine gland, and primary and secondary HFs. In silico analysis, reporter gene assays as well as in vitro transfections of LEF1 and beta-catenin show that Dkk4 is a potential downstream target of canonical Wnt signaling. In addition, we demonstrate a direct physical interaction between LEF1/beta-catenin complex and the Dkk4 promoter using ChIP. We propose that Dkk4 acts in a negative feedback loop to attenuate canonical Wnt signaling, and may facilitate a switch to the non-canonical Wnt planar cell polarity (PCP) pathway that is involved in cell movements during morphogenesis.

The Wnt inhibitor, Dickkopf 4, is induced by canonical Wnt signaling during ectodermal appendage morphogenesis.

The increasing use of the hair follicle as a stem cell paradigm is due in part to the complex interplay between epithelial, dermal and other cell types, each with interesting differentiation potential and prospective therapeutic applications. This review focuses on research into the environmental niche, gene expression profiles and plasticity of hair follicle stem cell populations, where many recent advances have come about through novel technological and experimental approaches. We discuss major developmental pathways involved in the establishment and control of the epithelial stem cell niche, and evidence of plasticity between stem and transit amplifying cell populations.

Hair follicle stem cells.

The corneal epithelium is continuously replaced by epithelial stem cells located in the basal layer of the limbus, located at the margin of the cornea. Studying how the stem cell niche is established at the limbus during development of the eye may lead to better understanding and treatments for diseases associated with limbal deficiencies. Using two highly specific commercially available antibodies, K10 was consistently detected suprabasally throughout the developing limbal epithelium of late gestation (20.5 dpc) and neonatal rat corneas, with interrupted expression in adult rat limbal epithelium. RT-PCR confirmed K10 expression at the transcript level in embryonic, neonatal and adult rat eyes. We have identified a time point where early stages of limbal development may be facilitated by the suprabasal expression of K10.

Keratin 10 (K10) is expressed suprabasally throughout the limbus of embryonic and neonatal rat corneas, with interrupted expression in the adult limbus.

Syndecan-1 is a cell-surface heparan-sulphate proteoglycan that is involved in growth factor regulation, cell adhesion, proliferation, differentiation, blood coagulation, lipid metabolism, as well as tumour formation. In this study, investigation of discrete LCM captured dermal cells by semi-quantitative RT-PCR revealed Syndecan-1 mRNA transcripts were expressed only in the dermal condensation (DC) within this skin compartment during murine pelage hair follicle (HF) morphogenesis. Further immunofluorescence studies showed that, during early skin development, Syndecan-1 was expressed in the epidermis while being absent from the mesenchyme. As HF morphogenesis began ( approximately E14.5) Syndecan-1 expression was lost from the epithelial compartment of the HF and activated in HF mesenchymal cells. This Syndecan-1 expression profile was consistent between different hair follicle types including primary and secondary pelage, vibrissa, and tail hair follicles. Furthermore we show by using gene targeted mice lacking Syndecan-1 expression that Syndecan-1 is not required for follicle initiation and development.

Dynamic expression of Syndecan-1 during hair follicle morphogenesis.

A key initial event in hair follicle morphogenesis is the localised thickening of the skin epithelium to form a placode, partitioning future hair follicle epithelium from interfollicular epidermis. Although many developmental signalling pathways are implicated in follicle morphogenesis, the role of epidermal growth factor (EGF) and keratinocyte growth factor (KGF, also known as FGF7) receptors are not defined. EGF receptor (EGFR) ligands have previously been shown to inhibit developing hair follicles; however, the underlying mechanisms have not been characterised. Here we show that receptors for EGF and KGF undergo marked downregulation in hair follicle placodes from multiple body sites, whereas the expression of endogenous ligands persist throughout hair follicle initiation. Using embryonic skin organ culture, we show that when skin from the sites of primary pelage and whisker follicle development is exposed to increased levels of two ectopic EGFR ligands (HBEGF and amphiregulin) and the FGFR2(IIIb) receptor ligand KGF, follicle formation is inhibited in a time- and dose-dependent manner. We then used downstream molecular markers and microarray profiling to provide evidence that, in response to KGF and EGF signalling, epidermal differentiation is promoted at the expense of hair follicle fate. We propose that hair follicle initiation in placodes requires downregulation of the two pathways in question, both of which are crucial for the ongoing development of the interfollicular epidermis. We have also uncovered a previously unrecognised role for KGF signalling in the formation of hair follicles in the mouse.

KGF and EGF signalling block hair follicle induction and promote interfollicular epidermal fate in developing mouse skin.

Exogen is a distinct phase of the hair cycle describing the process by which the hair club fibre is shed from the follicle. This process is difficult to study in human skin and little is known about the mechanisms involved in the release of club fibres. We sought an alternative model system to study exogen in more detail, and therefore utilised the vibrissa system on the rodent mystacial pad. The time at which a vibrissa club hair will be lost can be predicted, based on the relative lengths of the new growing fibre and old club fibre. This timing phenomenon was exploited to investigate the club fibre within the follicle as it approaches final release, revealing key changes in the adhesive state of the club fibre within the epithelial sac as it approached release. We propose that exogen should be subdivided to represent variations in the club fibre status.

Exogen involves gradual release of the hair club fibre in the vibrissa follicle model.

Human dermal papilla (DP) cells grown in two-dimensional (2D) culture have been studied extensively. However, key differences exist between DP cell activities in vivo and in vitro. Using a suspension method of cell culture to maintain DP cells, we created three-dimensional (3D) dermal spheres morphologically akin to intact (anagen) DPs. Analysis of these spheres using immunocytochemistry demonstrates that they have expression profiles different from papilla cells cultured in 2D but with many similarities to intact DPs. This method of DP cell culture may provide us with a tool to elucidate our understanding of signalling within the DP as it relates to induction, maintenance or even inhibition of hair growth.

Modelling the hair follicle dermal papilla using spheroid cell cultures.

Reprogramming of human hair follicle dermal papilla cells into induced pluripotent stem cells.

In this study, we have demonstrated that cells of neural crest origin located in the dermal papilla (DP) exhibit endothelial marker expression and a functional activity. When grown in endothelial growth media, DP primary cultures upregulate expression of vascular endothelial growth factor receptor 1 (FLT1) mRNA and downregulate expression of the dermal stem cell marker α-smooth muscle actin. DP cells have demonstrated functional characteristics of endothelial cells, including the ability to form capillary-like structures on Matrigel, increase uptake of low-density lipoprotein and upregulate ICAM1 (CD54) in response to tumour necrosis factor alpha (TNF-α) stimulation. We confirmed that these observations were not due to contaminating endothelial cells, by using DP clones. We have also used the WNT1cre/ROSA26R and WNT1cre/YFP lineage-tracing mouse models to identify a population of neural crest-derived cells in DP cultures that express the endothelial marker PECAM (CD31); these cells also form capillary-like structures on Matrigel. Importantly, cells of neural crest origin that express markers of endothelial and mesenchymal lineages exist within the dermal sheath of the vibrissae follicle.

Dermal stem cells can differentiate down an endothelial lineage.

While the mammalian heart has low, but functionally significant, levels of telomerase expression, the cellular population responsible remains incompletely characterized. This study aimed to identify the cell types responsible for cardiac telomerase activity in neonatal, adult, and cryoinjured adult hearts using transgenic mice expressing green fluorescent protein (GFP), driven by the promoter for murine telomerase reverse transcriptase (mTert), which is a necessary and rate-limiting component of telomerase. A rare population of mTert-GFP-expressing cells was identified that possessed all detectable cardiac telomerase RNA and telomerase activity. It was heterogeneous and included cells coexpressing markers of cardiomyocytic, endothelial, and mesenchymal lineages, putative cardiac stem cell markers, and, interestingly, cardiomyocytes with a differentiated phenotype. Quantification using both flow cytometry and immunofluorescence identified a significant decline in mTert-GFP cells in adult animals compared to neonates (∼9- and ∼20-fold, respectively). Cardiac injury resulted in a ∼6.45-fold expansion of this population (P<0.005) compared with sham-operated controls. This study identifies the cells responsible for cardiac telomerase activity, demonstrates a significant diminution with age but a marked response to injury, and, given the relationship between telomerase activity and stem cell populations, suggests that they represent a potential target for further investigation of cardiac regenerative potential.

Telomerase expression in the mammalian heart.

Cardiac side population (CSP) cells, defined by their ability to efflux the vital dye Hoechst 33342, have been identified as putative cardiac stem cells based on their potential to give rise to both cardiomyocytes and endothelial cells. The CSP phenotype relies on an active metabolic pathway and cell viability to identify a rare population of cells and therefore technical differences in the CSP staining protocol can lead to inconsistent results and discrepancies between studies. Here we describe an established protocol for CSP identification and have optimised a protocol for CSP analysis utilizing an automated cardiac digestion procedure using gentleMACs dissociation and Hoechst 33342 staining followed by dual wavelength flow cytometric analysis.

Article	Year
CD133, a novel marker for human prostatic epithelial stem cells. Journal of cell science\| PubMed ID: 15226377	2004
Cultured cells from the adult human hair follicle dermis can be directed toward adipogenic and osteogenic differentiation. The Journal of investigative dermatology\| PubMed ID: 15854060	2005
Plasticity of rodent and human hair follicle dermal cells: implications for cell therapy and tissue engineering. The journal of investigative dermatology. Symposium proceedings / the Society for Investigative Dermatology, Inc. [and] European Society for Dermatological Research\| PubMed ID: 16382659	2005
Transcriptional profiling of developing mouse epidermis reveals novel patterns of coordinated gene expression. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 17330888	2007
The Wnt inhibitor, Dickkopf 4, is induced by canonical Wnt signaling during ectodermal appendage morphogenesis. Developmental biology\| PubMed ID: 17397822	2007
Hair follicle stem cells. Seminars in cell & developmental biology\| PubMed ID: 17481931	2007
Keratin 10 (K10) is expressed suprabasally throughout the limbus of embryonic and neonatal rat corneas, with interrupted expression in the adult limbus. Experimental eye research\| PubMed ID: 19344714	2009
Dynamic expression of Syndecan-1 during hair follicle morphogenesis. Gene expression patterns : GEP\| PubMed ID: 19427408	2009
KGF and EGF signalling block hair follicle induction and promote interfollicular epidermal fate in developing mouse skin. Development (Cambridge, England)\| PubMed ID: 19474150	2009
Exogen involves gradual release of the hair club fibre in the vibrissa follicle model. Experimental dermatology\| PubMed ID: 19695019	2009
Modelling the hair follicle dermal papilla using spheroid cell cultures. Experimental dermatology\| PubMed ID: 20456497	2010
Reprogramming of human hair follicle dermal papilla cells into induced pluripotent stem cells. The Journal of investigative dermatology\| PubMed ID: 22336943	2012
Dermal stem cells can differentiate down an endothelial lineage. Stem cells and development\| PubMed ID: 22571645	2012
Telomerase expression in the mammalian heart. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 22919071	2012
Optimised protocols for the identification of the murine cardiac side population. Stem cell reviews\| PubMed ID: 23619929	2013

Gavin D. Richardson

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