TGFß signaling underlies hematopoietic dysfunction and bone marrow failure in Shwachman-Diamond Syndrome.

Shwachman-Diamond Syndrome (SDS) is a rare and clinically-heterogeneous bone marrow (BM) failure syndrome caused by mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene. Although SDS was described over 50 years ago, the molecular pathogenesis is poorly understood due, in part, to the rarity and heterogeneity of the affected hematopoietic progenitors. To address this, we used single cell RNA sequencing to profile scant hematopoietic stem and progenitor cells from SDS patients. We generated a single cell map of early lineage commitment and found that SDS hematopoiesis was left-shifted with selective loss of granulocyte-monocyte progenitors. Transcriptional targets of transforming growth factor-beta (TGFß) were dysregulated in SDS hematopoietic stem cells and multipotent progenitors, but not in lineage-committed progenitors. TGFß inhibitors (AVID200 and SD208) increased hematopoietic colony formation of SDS patient BM. Finally, TGFß3 and other TGFß pathway members were elevated in SDS patient blood plasma. These data establish the TGFß pathway as a novel candidate biomarker and therapeutic target in SDS and translate insights from single cell biology into a potential therapy.

The lncRNA SLNCR Recruits the Androgen Receptor to EGR1-Bound Genes in Melanoma and Inhibits Expression of Tumor Suppressor p21.

Melanoma is the deadliest form of skin cancer, affecting men more frequently and severely than women. Although recent studies suggest that differences in activity of the androgen receptor (AR) underlie the observed sex bias, little is known about AR activity in melanoma. Here we show that AR and EGR1 bind to the long non-coding RNA SLNCR and increase melanoma proliferation through coordinated transcriptional regulation of several growth-regulatory genes. ChIP-seq reveals that ligand-free AR is enriched on SLNCR-regulated melanoma genes and that AR genomic occupancy significantly overlaps with EGR1 at consensus EGR1 binding sites. We present a model in which SLNCR recruits AR to EGR1-bound genomic loci and switches EGR1-mediated transcriptional activation to repression of the tumor suppressor p21Waf1/Cip1. Our data implicate the regulatory triad of SLNCR, AR, and EGR1 in promoting oncogenesis and may help explain why men have a higher incidence of and more rapidly progressive melanomas compared with women.

Insulin-like growth factor 2 modulates murine hematopoietic stem cell maintenance through upregulation of p57.

Hematopoietic stem cells (HSC) rely on a highly regulated molecular network to balance self-renewal and lineage specification to sustain life-long hematopoiesis. Despite a plethora of studies aimed at identifying molecules governing HSC fate, our current knowledge of the genes responsible is limited. We have found insulin-like growth factor 2 (IGF2) to be expressed predominantly within long-term HSCs. This study examines IGF2 expression patterns and the effects of the gene in HSCs. Through the overexpression and knockdown of IGF2 within purified HSCs, we report that IGF2 expression increases HSC-derived multilineage colonies in vitro and enhances hematopoietic contribution in vivo on competitive bone marrow transplantation. The effects of IGF2 are mediated by direct upregulation of the CDKi p57, exclusively within long-term HSCs, via activation of the PI3K-Akt pathway. Increased expression of p57 resulted in a concomitant increase in HSCs in the G0/G1 stage of the cell cycle. Analysis of genomic DNA methylation revealed that HSCs exhibited a hypomethylated state within the promoter region of the CDKN1C (p57) gene, providing a potential mechanism for the exclusive effects of IGF2 within HSCs. Our studies indicate a novel role for IGF2 in regulating HSC cell cycle and illustrate potential novel therapeutic targets for hematologic diseases.

Efficient transduction of hematopoietic stem cells and its potential for gene correction of hematopoietic diseases.

The ability to efficiently transduce hematopoietic stem cells (HSC) represents a powerful methodology by which to study the role of specific genes on HSC function, as well as to broaden the potential of gene therapy for hematopoietic related diseases. While retroviruses have been used extensively to transduce a variety of cell types, HIV-derived lentiviruses prove superior for transduction of quiescent HSC due to their ability to infect non-dividing cells. Quality of lentiviral supernatants and starting cells are vital to obtain reproducible consistent results, and therefore, here we describe the production of concentrated lentiviral preparations, the purification of HSC from total mouse bone marrow, and their transduction to obtain long-term HSC engraftment with persistent gene transfer and expression of the desired transgene.

Excision of reprogramming transgenes improves the differentiation potential of iPS cells generated with a single excisable vector.

The residual presence of integrated transgenes following the derivation of induced pluripotent stem (iPS) cells is highly undesirable. Here we demonstrate efficient derivation of iPS cells free of exogenous reprogramming transgenes using an excisable polycistronic lentiviral vector. A novel version of this vector containing a reporter fluorochrome allows direct visualization of vector excision in living iPS cells in real time. We find that removal of the reprogramming vector markedly improves the developmental potential of iPS cells and significantly augments their capacity to undergo directed differentiation in vitro. We further propose that methods to efficiently excise reprogramming transgenes with minimal culture passaging, such as those demonstrated here, are critical since we find that iPS cells may acquire chromosomal abnormalities, such as trisomy of chromosome 8, similar to embryonic stem cells after expansion in culture. Our findings illustrate an efficient method for the generation of transgene-free iPS cells and emphasize the potential beneficial effects that may result from elimination of integrated reprogramming factors. In addition, our results underscore the consequences of long-term culture that will need to be taken into account for the clinical application of iPS cells.

CD133+ anaplastic thyroid cancer cells initiate tumors in immunodeficient mice and are regulated by thyrotropin.

BACKGROUND: Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies. Its rapid onset and resistance to conventional therapeutics contribute to a mean survival of six months after diagnosis and make the identification of thyroid-cancer-initiating cells increasingly important. METHODOLOGY/PRINCIPAL FINDINGS: In prior studies of ATC cell lines, CD133(+) cells exhibited stem-cell-like features such as high proliferation, self-renewal and colony-forming ability in vitro. Here we show that transplantation of CD133(+) cells, but not CD133(-) cells, into immunodeficient NOD/SCID mice is sufficient to induce growth of tumors in vivo. We also describe how the proportion of ATC cells that are CD133(+) increases dramatically over three months of culture, from 7% to more than 80% of the total. This CD133(+) cell pool can be further separated by flow cytometry into two distinct populations: CD133(+/high) and CD133(+/low). Although both subsets are capable of long-term tumorigenesis, the rapidly proliferating CD133(+/high) cells are by far the most efficient. They also express high levels of the stem cell antigen Oct4 and the receptor for thyroid stimulating hormone, TSHR. Treating ATC cells with TSH causes a three-fold increase in the numbers of CD133(+) cells and elicits a dose-dependent up-regulation of the expression of TSHR and Oct4 in these cells. More importantly, immunohistochemical analysis of tissue specimens from ATC patients indicates that CD133 is highly expressed on tumor cells but not on neighboring normal thyroid cells. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first report indicating that CD133(+) ATC cells are solely responsible for tumor growth in immunodeficient mice. Our data also give a unique insight into the regulation of CD133 by TSH. These highly tumorigenic CD133(+) cells and the activated TSH signaling pathway may be useful targets for future ATC therapies.

Thyroid stem cells: lessons from normal development and thyroid cancer.

Ongoing advances in stem cell research have opened new avenues for therapy for many human disorders. Until recently, however, thyroid stem cells have been relatively understudied. Here, we review what is known about thyroid stem cells and explore their utility as models of normal and malignant biological development. We also discuss the cellular origin of thyroid cancer stem cells and explore the clinical implications of cancer stem cells in the thyroid gland. Since thyroid cancer is the most common form of endocrine cancer and that thyroid hormone is needed for the growth and metabolism of each cell in the body, understanding the molecular and the cellular aspects of thyroid stem cell biology will ultimately provide insights into mechanisms underlying human disease.

Haptide-coated collagen sponge as a bioactive matrix for tissue regeneration.

We previously described a new class of conserved, cell adhesive (haptotactic) peptides, termed Haptides, based on sequences first identified in fibrinogen. Here, we describe a new biomaterial, Haptide-coated Collagen, in which the carbodiimide reagent, EDC, was used to covalently couple a Haptide (preC gamma), equivalent to the carboxy terminus of the fibrinogen gamma chain, to a cross-linked sponge composed of bovine collagen type I. The dose response of Haptide bound to collagen on cell attachment response reached a plateau at a concentration of 5-10 mg Haptide/g collagen. The Haptized-collagen was more stable to 1N NaOH, with a degradation half-time (T(1/2)) of 1.7 h, compared to 0.9 h for untreated control. Haptized collagen discs could be loaded with approximately 30% more human dermal fibroblasts or bovine aortic endothelial cells than unmodified collagen discs (p < 0.001). After a proliferation phase, Haptized collagen discs contained approximately 45% more fibroblasts than non-Haptized discs (p < 0.01). Histological analysis following sub-dermal implantation in rats indicated that at day 8, Haptized collagen sponge was less degraded than unmodified collagen sponge, attracted more endogenous fibroblasts with newly deposited collagen, and provoked less inflammatory or other adverse reactions. These results suggest potential clinical applications for Haptized collagen sponge for tissue regeneration, soft tissue augmentation, skin repair, and wound healing.