Karen B. Chapman

Spontaneous reversal of the developmental aging of normal human cells following transcriptional reprogramming

AIM To determine whether transcriptional reprogramming is capable of reversing the developmental aging of normal human somatic cells to an embryonic state. MATERIALS & METHODS An isogenic system was utilized to facilitate an accurate assessment of the reprogramming of telomere restriction fragment (TRF) length of aged differentiated cells to that of the human embryonic stem (hES) cell line from which they were originally derived. An hES-derived mortal clonal cell strain EN13 was reprogrammed by SOX2, OCT4 and KLF4. The six resulting induced pluripotent stem (iPS) cell lines were surveyed for telomere length, telomerase activity and telomere-related gene expression. In addition, we measured all these parameters in widely-used hES and iPS cell lines and compared the results to those obtained in the six new isogenic iPS cell lines. RESULTS We observed variable but relatively long TRF lengths in three widely studied hES cell lines (16.09-21.1 kb) but markedly shorter TRF lengths (6.4-12.6 kb) in five similarly widely studied iPS cell lines. Transcriptome analysis comparing these hES and iPS cell lines showed modest variation in a small subset of genes implicated in telomere length regulation. However, iPS cell lines consistently showed reduced levels of telomerase activity compared with hES cell lines. In order to verify these results in an isogenic background, we generated six iPS cell clones from the hES-derived cell line EN13. These iPS cell clones showed initial telomere lengths comparable to the parental EN13 cells, had telomerase activity, expressed embryonic stem cell markers and had a telomere-related transcriptome similar to hES cells. Subsequent culture of five out of six lines generally showed telomere shortening to lengths similar to that observed in the widely distributed iPS lines. However, the clone EH3, with relatively high levels of telomerase activity, progressively increased TRF length over 60 days of serial culture back to that of the parental hES cell line. CONCLUSION Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine.

The ACTCellerate initiative: large-scale combinatorial cloning of novel human embryonic stem cell derivatives

Human embryonic stem cells offer a scalable and renewable source of all somatic cell types. Human embryonic progenitor (hEP) cells are partially differentiated endodermal, mesodermal and ectodermal cell types that have not undergone terminal differentiation and express an embryonic pattern of gene expression. Here, we describe a large-scale and reproducible method of isolating a diverse library of clonally purified hEP cell lines, many of which are capable of extended propagation in vitro. Initial microarray and non-negative matrix factorization gene-expression profiling suggests that the library consists of at least 140 distinct clones and contains many previously uncharacterized cell types derived from all germ layers that display diverse embryo- and site-specific homeobox gene expression. Despite the expression of many oncofetal genes, none of the hEP cell lines tested led to tumor formation when transplanted into immunocompromised mice. All hEP lines studied appear to have a finite replicative lifespan but have longer telomeres than most fetal- or adult-derived cells, thereby facilitating their use in the manufacture of purified lineages for research and human therapy.

COL10A1 expression is elevated in diverse solid tumor types and is associated with tumor vasculature

AIM The identification of molecular markers that are upregulated in multiple tumor types could lead to novel diagnostic and therapeutic strategies. The authors screened a panel of RNAs prepared from diverse tumors and tumor cell lines, and compared them with normal tissues and cultured somatic cell types, in order to identify candidate genes expressed in a broad spectrum of tumor types. MATERIALS & METHODS Gene expression microarray analysis was carried out on 128 individual tumor samples representing over 20 tumor types, 85 samples representing 31 diverse normal tissue types, 68 tumor cell lines and 97 diverse normal primary cell cultures. Genes were ranked for elevated expression across a large number and variety of tumors relative to normal tissues. RESULTS & CONCLUSION COL10A1 was identified as a gene with restricted expression in most normal tissues and elevated expression in many diverse tumor types. By contrast, COL10A1 expression was undetectable in the 68 tumor cell lines surveyed in this study. Immunofluorescence studies localized collagen, type X, α-1 (collagen X) staining to tumor vasculature in breast tumors, whereas the vasculature of normal breast tissue was either collagen X-negative or had markedly lower levels. The tumor microenvironment-specific expression of collagen X, together with its localization in the vasculature, may facilitate its use as a novel target for the diagnosis and treatment of diverse solid tumor types.

A human embryonic stem cell-derived clonal progenitor cell line with chondrogenic potential and markers of craniofacial mesenchyme

AIMS We screened 100 diverse human embryonic stem-derived progenitor cell lines to identify novel lines with chondrogenic potential. MATERIALS & METHODS The 4D20.8 cell line was compared with mesenchymal stem cells and dental pulp stem cells by assessing osteochondral markers using immunohistochemical methods, gene expression microarrays, quantitative real-time PCR and in vivo repair of rat articular condyles. RESULTS 4D20.8 expressed the site-specific gene markers LHX8 and BARX1 and robustly upregulated chondrocyte markers upon differentiation. Differentiated 4D20.8 cells expressed relatively low levels of COL10A1 and lacked IHH and CD74 expression. Transplantation of 4D20.8 cells into experimentally induced defects in the femoral condyle of athymic rats resulted in cartilage and bone differentiation approximating that of the original tissue architecture. Relatively high COL2A1 and minimal COL10A1 expression occurred during differentiation in HyStem-C hydrogel with TGF-β3 and GDF-5. CONCLUSION Human embryonic stem cell-derived embryonic progenitor cell lines may provide a novel means of generating purified site-specific osteochondral progenitor cell lines that are useful in research and therapy.

Seven diverse human embryonic stem cell-derived chondrogenic clonal embryonic progenitor cell lines display site-specific cell fates

AIM The transcriptomes of seven diverse clonal human embryonic progenitor cell lines with chondrogenic potential were compared with that of bone marrow-derived mesenchymal stem cells (MSCs). MATERIALS & METHODS The cell lines 4D20.8, 7PEND24, 7SMOO32, E15, MEL2, SK11 and SM30 were compared with MSCs using immunohistochemical methods, gene expression microarrays and quantitative real-time PCR. RESULTS In the undifferentiated progenitor state, each line displayed unique combinations of site-specific markers, including AJAP1, ALDH1A2, BMP5, BARX1, HAND2, HOXB2, LHX1, LHX8, PITX1, TBX15 and ZIC2, but none of the lines expressed the MSC marker CD74. The lines showed diverse responses when differentiated in the presence of combinations of TGF-β3, BMP2, 4, 6 and 7 and GDF5, with the lines 4D20.8, SK11, SM30 and MEL2 showing osteogenic markers in some differentiation conditions. The line 7PEND24 showed evidence of regenerating articular cartilage and, in some conditions, markers of tendon differentiation. CONCLUSION The scalability of site-specific clonal human embryonic stem cell-derived embryonic progenitor cell lines may provide novel models for the study of differentiation and methods for preparing purified and identified cells types for use in therapy.

Elevated expression of cancer/testis antigen FSIP1 in ER-positive breast tumors.

AIM The study aimed to identify and characterize highly specific breast tumor biomarkers. METHODS A microarray data set comprised of 513 diverse normal and tumor mRNA samples was analyzed to identify breast tumor biomarkers with minimal expression in normal tissues. RESULTS FSIP1 was identified as a breast tumor biomarker with elevated mRNA expression in breast tumors and minimal expression in most normal tissues except the testis. Quantitative real-time PCR confirmed the elevated expression of FSIP1 mRNA in breast tumors and revealed a significant correlation with ER-positive status. Immunofluorescence staining of breast tumor sections showed that the majority of breast tumors examined in this study (20 out of 22) expressed detectable FSIP1 protein, with significantly higher than average expression in ER-positive versus ER-negative breast tumors. CONCLUSION The prevalence and uniformity of FSIP1 expression in breast tumors, taken together with the highly restricted expression in normal tissues, suggests that FSIP1 may be an attractive target for breast cancer immunotherapy.

Human embryonic stem cell-derived neural crest cells capable of expressing markers of osteochondral or meningeal-choroid plexus differentiation

AIMS The transcriptome and fate potential of three diverse human embryonic stem cell-derived clonal embryonic progenitor cell lines with markers of cephalic neural crest are compared when differentiated in the presence of combinations of TGFβ3, BMP4, SCF and HyStem-C matrices. MATERIALS & METHODS The cell lines E69 and T42 were compared with MEL2, using gene expression microarrays, immunocytochemistry and ELISA. RESULTS In the undifferentiated progenitor state, each line displayed unique markers of cranial neural crest including TFAP2A and CD24; however, none expressed distal HOX genes including HOXA2 or HOXB2, or the mesenchymal stem cell marker CD74. The lines also showed diverse responses when differentiated in the presence of exogenous BMP4, BMP4 and TGFβ3, SCF, and SCF and TGFβ3. The clones E69 and T42 showed a profound capacity for expression of endochondral ossification markers when differentiated in the presence of BMP4 and TGFβ3, choroid plexus markers in the presence of BMP4 alone, and leptomeningeal markers when differentiated in SCF without TGFβ3. CONCLUSION The clones E69 and T42 may represent a scalable source of primitive cranial neural crest cells useful in the study of cranial embryology, and potentially cell-based therapy.

Adult Versus Pluripotent Stem Cell-Derived Mesenchymal Stem Cells: The Need for More Precise Nomenclature

The complexity of human pluripotent stem cell (hPSC) fate represents both opportunity and challenge. In theory, all somatic cell types can be differentiated from hPSCs, opening the door to many opportunities in transplant medicine. However, such clinical applications require high standards of purity and identity, that challenge many existing protocols. This underscores the need for increasing precision in the description of cell identity during hPSC differentiation. We highlight one salient example, namely, the numerous published reports of hPSC-derived mesenchymal stem cells (MSCs). We suggest that many of these reports likely represent an improper use of certain cluster of differentiation (CD) antigens in defining bone marrow-derived MSCs. Instead, most such hPSC-derived mesenchymal cells are likely a complex mixture of embryonic anlagen, primarily of diverse mesodermal and neural crest origins, making precise identification, reproducible manufacture, and uniform differentiation difficult to achieve. We describe a potential path forward that may provide more precision in nomenclature, and cells with higher purity and identity for potential therapeutic use.

The germline/soma dichotomy: implications for aging and degenerative disease

Human somatic cells are mortal due in large part to telomere shortening associated with cell division. Limited proliferative capacity may, in turn, limit response to injury and may play an important role in the etiology of age-related pathology. Pluripotent stem cells cultured in vitro appear to maintain long telomere length through relatively high levels of telomerase activity. We propose that the induced reversal of cell aging by transcriptional reprogramming, or alternatively, human embryonic stem cells engineered to escape immune surveillance, are effective platforms for the industrial-scale manufacture of young cells for the treatment of age-related pathologies. Such cell-based regenerative therapies will require newer manufacturing and delivery technologies to insure highly pure, identified and potent pluripotency-based therapeutic formulations.

Abstract 551: Identification of gene-expression biomarkers in urine pathology specimens for the detection of bladder cancer

Bladder cancer recurrence screening is typically managed with a combination of urine cytology and cystoscopy, each with its inherent caveats. Cystoscopy is generally considered the gold standard for diagnosing bladder cancer and can yield a valuable biopsy, but is too invasive and expensive for routine screening and surveillance. Voided urine cytology is inexpensive and readily available, but lacks the desired level of sensitivity. Furthermore, up to 20% of urine cytology specimens fall into one of two indeterminate categories that require follow-up testing, typically in the form of an invasive cystoscopy procedure. With the goal of developing a urine-based molecular test for bladder cancer recurrence surveillance, we examined gene expression biomarkers in urine cytopathology specimens. 90 patient urine samples were analyzed: 45 high-grade urothelial carcinoma (HG-UC) and 45 benign samples. All patient samples were submitted to the Johns Hopkins Cytopathology Department for evaluation for bladder cancer via urine cytology. The samples represent patients undergoing bladder cancer recurrence screening and patients without a prior history of bladder cancer presenting with hematuria. The urine sediments were stored in liquid based cytology solution at 4°C for a period of 7 - 10 days, during which time the cytopathology diagnosis was determined from a portion of the sample. Post-diagnosis, the pathology sample “leftover” was analyzed for gene expression. RNA was extracted, amplified and microarray analysis was performed and a panel of molecular biomarkers was identified. Individual genes in this panel discriminate between HG-UC and benign in this training set with an average ROC of 0.86 (p Citation Format: Karen B. Chapman, Liqun Qiu, Jennifer Kidd, Aparna Baxi, Markus D. Lachter, Joseph Wagner, Dorothy L. Rosenthal, Matthew T. Olson. Identification of gene-expression biomarkers in urine pathology specimens for the detection of bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 551. doi:10.1158/1538-7445.AM2015-551