Extracellular vesicles from mesenchymal stem cells of dental pulp and adipose tissue display distinct transcriptomic characteristics suggestive of potential therapeutic targets.

Mesenchymal stem cells (MSCs) are isolated from various human tissues and used for therapy, in which beneficial effects are attributed mainly to mesenchymal stem cell-derived extracellular vesicles (MSC-EVs). Whereas MSCs of diverse tissue types share cardinal stem cell features, it is becoming evident that MSCs of each tissue type possess unique properties as well. For designing efficient stem cellbased therapies, it is crucial to understand the unique properties associated with MSCs and MSC-EVs of each tissue type.

Comparative transcriptomic analysis of human mesenchymal stem cells derived from dental pulp and adipose tissues.

Mesenchymal stem cells (MSCs) have been isolated from various human tissues. Although they share cardinal stem cell features of self-renewal and multi-potency, they also seem to possess distinct characteristics depending on the tissue types they originated from. When developing stem cell-based therapies, MSCs with the most desirable characteristics should be chosen.

Metabolic profiles are principally different between cancers of the liver, pancreas and breast.

Molecular profiling of primary tumors may facilitate the classification of patients with cancer into more homogenous biological groups to aid clinical management. Metabolomic profiling has been shown to be a powerful tool in characterizing the biological mechanisms underlying a disease but has not been evaluated for its ability to classify cancers by their tissue of origin. Thus, we assessed metabolomic profiling as a novel tool for multiclass cancer characterization.

Ex vivo-expanded natural killer cells kill cancer cells more effectively than ex vivo-expanded γδ T cells or αβ T cells.

Adoptive immunotherapy of cancer is evolving with the development of novel technologies for generating a large number of activated killer cells such as natural killer (NK) cells, γδ T cells, and αβ T cells. We have recently established large-scale culture methods to generate activated NK cells from human peripheral blood, and demonstrated that expanded NK cells have higher cytotoxicity against cancer cells than freshly isolated NK cells. In this study, we compared cultured NK cells with cultured γδ T and αβ T cells that were prepared by conventional culture methods regarding the expression of cytotoxic molecules and cytotoxicity against cancer cells.

Pathway-centric integrative analysis identifies RRM2 as a prognostic marker in breast cancer associated with poor survival and tamoxifen resistance.

Breast cancer (BCa) molecular subtypes include luminal A, luminal B, normal-like, HER-2-enriched, and basal-like tumors, among which luminal B and basal-like cancers are highly aggressive. Biochemical pathways associated with patient survival or treatment response in these more aggressive subtypes are not well understood. With the limited availability of pathologically verified clinical specimens, cell line models are routinely used for pathway-centric studies.

MYC-driven accumulation of 2-hydroxyglutarate is associated with breast cancer prognosis.

Metabolic profiling of cancer cells has recently been established as a promising tool for the development of therapies and identification of cancer biomarkers. Here we characterized the metabolomic profile of human breast tumors and uncovered intrinsic metabolite signatures in these tumors using an untargeted discovery approach and validation of key metabolites. The oncometabolite 2-hydroxyglutarate (2HG) accumulated at high levels in a subset of tumors and human breast cancer cell lines.

Perivascular hair follicle stem cells associate with a venule annulus.

The perivascular microenvironment helps in maintaining stem cells in many tissues. We sought to determine whether there is a perivascular niche for hair follicle stem cells. The association of vessels and follicle progenitor cells began by embryonic day 14.

Interferon regulatory factor 8 integrates T-cell receptor and cytokine-signaling pathways and drives effector differentiation of CD8 T cells.

We recently demonstrated that differentiation of cytotoxic T cells requires cooperation between T-cell receptor (TCR)/costimulation and γc-cytokines. Here we demonstrate that the transcription factor IFN regulatory factor 8 (IRF8) is expressed in CD8 T cells by the combination of these two signals. More importantly, depletion of IRF8 in these cells abrogated the differentiation of naive CD8 T cells into effector cells in an experimental graft-vs.

Relationship between tumor DNA methylation status and patient characteristics in African-American and European-American women with breast cancer.

Aberrant DNA methylation is critical for development and progression of breast cancer. We investigated the association of CpG island methylation in candidate genes and clinicopathological features in 65 African-American (AA) and European-American (EA) breast cancer patients. Quantitative methylation analysis was carried out on bisulfite modified genomic DNA and sequencing (pyrosequencing) for promoter CpG islands of p16, ESR1, RASSF1A, RARβ2, CDH13, HIN1, SFRP1 genes and the LINE1 repetitive element using matched paired non-cancerous and breast tumor specimen (32 AA and 33 EA women).

Identification and characterization of tumor-initiating cells in human primary cutaneous squamous cell carcinoma.

Primary human squamous cell carcinomas (SCCas) are heterogeneous invasive tumors with proliferating outer layers and inner differentiating cell masses. To determine if tumor-initiating cells (TICs) are present in SCCas, we utilized newly developed reliable in vitro and in vivo xenograft assays that propagate human SCCas, and demonstrated that a small subset of SCCa cells (∼1%) expressing Prominin-1 (CD133) in the outer layers of SCCas were highly enriched for TICs (∼1/400) compared with unsorted SCCa cells (TICs ∼1/10(6)). Xenografts of CD133+ SCCas recreated the original SCCa tumor histology and organizational hierarchy, whereas CD133- cells did not, and only CD133+ cells demonstrated the capacity for self-renewal in serial transplantation studies.

A gene therapy approach for long-term normalization of blood pressure in hypertensive mice by ANP-secreting human skin grafts.

The use of bioengineered human skin as a bioreactor to deliver therapeutic factors has a number of advantages including accessibility that allows manipulation and monitoring of genetically modified cells. We demonstrate a skin gene therapy approach that can regulate blood pressure and treat systemic hypertension by expressing atrial natriuretic peptide (ANP), a hormone able to decrease blood pressure, in bioengineered human skin equivalents (HSE). Additionally, the expression of a selectable marker gene, multidrug resistance (MDR) type 1, is linked to ANP expression on a bicistronic vector and was coexpressed in the human keratinocytes and fibroblasts of the HSE that were grafted onto immunocompromised mice.

Efficient procurement of epithelial stem cells from human tissue specimens using a Rho-associated protein kinase inhibitor Y-27632.

The efficient culture of stem cells from epithelial tissues such as skin and corneas is important for both experimental studies and clinical applications of tissue engineering. We now demonstrate that treatment of human-skin-derived keratinocytes with a Rho-associated protein kinase inhibitor Y-27632 for the initial 6 days of primary culture can increase the number of keratinocytes that possess stem cell properties to form colonies during in vitro culture of freshly isolated cells and subsequent passage (50-fold). Further, we show that Y-27632 treatment can increase the total number of prostate epithelial cells derived from human prostate specimens.

Stem cell activity of human side population and alpha6 integrin-bright keratinocytes defined by a quantitative in vivo assay.

The isolation and characterization of living human epithelial stem cells is difficult because distinguishing cell surface markers have not been identified with certainty. Side population keratinocytes (SP-KCs) that efflux Hoechst 33342 fluorescent dye, analogous to bone marrow-derived side population (SP) hematopoietic stem cells, have been identified in human skin, but their potential to function as keratinocyte stem cells (KSCs) in vivo is not known. On the other hand, human keratinocyte populations that express elevated levels of beta1 and alpha6 integrins and are distinct from SP-KCs, which express low levels of integrins, may be enriched for KSCs based on reported results of in vitro cell culture assays.

Characterization and isolation of stem cell-enriched human hair follicle bulge cells.

The human hair follicle bulge is an important niche for keratinocyte stem cells (KSCs). Elucidation of human bulge cell biology could be facilitated by analysis of global gene expression profiles and identification of unique cell-surface markers. The lack of distinctive bulge morphology in human hair follicles has hampered studies of bulge cells and KSCs.

A proteomic characterization of the plasma membrane of human epidermis by high-throughput mass spectrometry.

Membrane proteins are responsible for many critical cellular functions and identifying cell surface proteins on different keratinocyte populations by proteomic approaches would improve our understanding of their biological function. The ability to characterize membrane proteins, however, has lagged behind that of soluble proteins both in terms of throughput and protein coverage. In this study, a membrane proteomic investigation of keratinocytes using a two-dimensional liquid chromatography (LC) tandem-mass spectrometry (MS/MS) approach that relies on a buffered methanol-based solubilization, and tryptic digestion of purified plasma membrane is described.

Ultraviolet light selection assay to optimize oligonucleotide correction of mutations in endogenous xeroderma pigmentosum genes.

Various oligonucleotide (ODN)-based approaches have been proposed for their ability to correct mutated genes at the normal chromosomal locations. However, the reported gene correction frequencies of these approaches have varied markedly in different experimental settings, including when different tissues or cell types are targeted. In order to find the optimal ODN-based approach for a specific target tissue, an assay system that allows direct comparison of the different methods on that tissue is necessary.

Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells.

Very primitive hematopoietic stem cells have been identified as side population cells based on their ability to efflux a fluorescent vital dye, Hoechst 33342. In this study we show that keratinocytes with the same side population phenotype are also present in the human epidermis. Although side population keratinocytes have the same dye-effluxing phenotype as bone marrow side population cells and can be blocked by verapamil, they do not express increased levels of the ABCG2 transporter that is believed to be responsible for the bone marrow side population phenotype.

Quantitative proteomics employing primary amine affinity tags.

A proteomics-based method using stable isotope labeling to assess the relative abundance of peptides or proteins is described. Bradykinin and carbonic anhydrase were labeled with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate, a membrane impermeant reagent that is reactive with primary amines. Specificity of the label to primary amines was demonstrated using tandem mass spectrometry.

Topical colchicine selection of keratinocytes transduced with the multidrug resistance gene (MDR1) can sustain and enhance transgene expression in vivo.

For skin gene therapy, achieving prolonged high-level gene expression in a significant percentage of keratinocytes (KC) is difficult because we cannot selectively target KC stem cells. We now demonstrate that topical colchicine treatment can be used to select, in vivo, KC progenitor cells transduced with the multidrug resistance gene (MDR1). When human skin equivalents containing MDR1-transduced KC were grafted onto immunocompromised mice, topical colchicine treatments significantly increased (7-fold) the percentage of KC expressing MDR1, compared to vehicle-treated controls, for up to 24 wk.

In vivo assessment of gene delivery to keratinocytes by lentiviral vectors.

For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction.