Hal Sternberg

Umbilical Cord Tissue Offers the Greatest Number of Harvestable Mesenchymal Stem Cells for Research and Clinical Application: A Literature Review of Different Harvest Sites

PURPOSE Recent years have seen dramatic increases in the techniques used to harvest and isolate human mesenchymal stem cells. As the potential therapeutic aspects of these cells further develop, informative data on the differences in yields between tissue harvest sites and methods will become increasingly valuable. We collected and compared data on cell yields from multiple tissue harvest sites to provide insight into the varying levels of mesenchymal stem cells by tissue and offer primary and alternative tissue types for harvest and clinical application. METHODS The PubMed and Medline databases were searched for articles relating to the harvest, isolation, and quantification of human mesenchymal stem cells. Selected articles were analyzed for relevant data, which were categorized according to tissue site and, if possible, standardized to facilitate comparison between sites. RESULTS Human mesenchymal stem cell levels in tissue varied widely according to tissue site and harvest method. Yields for adipose tissue ranged from 4,737 cells/mL of tissue to 1,550,000 cells/mL of tissue. Yields for bone marrow ranged from 1 to 30 cells/mL to 317,400 cells/mL. Yields for umbilical cord tissue ranged from 10,000 cells/mL to 4,700,000 cells/cm of umbilical cord. Secondary tissue harvest sites such as placental tissue and synovium yielded results ranging from 1,000 cells/mL to 30,000 cells/mL. CONCLUSIONS Variations in allogeneic mesenchymal stem cell harvest levels from human tissues reflect the evolving nature of the field, patient demographic characteristics, and differences in harvest and isolation techniques. At present, Whartons jelly tissue yields the highest concentration of allogeneic mesenchymal stem cells whereas adipose tissue yields the highest levels of autologous mesenchymal stem cells per milliliter of tissue. CLINICAL RELEVANCE This comparison of stem cell levels from the literature offers a primer and guide for harvesting mesenchymal stem cells. Larger mesenchymal stem cell yields are more desirable for research and clinical application.

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.

Identification of human embryonic progenitor cell targeting peptides using phage display.

Human pluripotent stem (hPS) cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS) cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES) cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken together these data suggest that selection of phage display libraries against a clonal progenitor stem cell population can be used to identify progenitor stem cell targeting peptides. The peptides may be useful for monitoring hPS cell differentiation and for the development of cell enrichment procedures to improve the efficiency of directed differentiation toward clinically relevant human cell types.

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.

Age-dependent loss of insulin-like growth factor-1 receptor immunoreactive cells in the supraoptic hypothalamus is reduced in calorically restricted mice.

Both life‐long caloric restriction (CR) and the suppression of insulin‐like growth factor‐1 (IGF‐1) signaling reliably extend the mammalian lifespan. The neuroendocrine system, regulated by the hypothalamus, remains the most convincing site of action for both these modes of life extension. Yet, determining whether CR actions are mediated by the modulation of neuroendocrine IGF‐1 signaling remains unclear. Of the hypothalamic nuclei that express the IGF‐1 receptor (IGF‐1R), the cells of the supraoptic nucleus (SON) display some of the most robust IGF‐1R expression. Taking IGF‐1R immunoreactivity as an index of sensitivity to IGF‐1, we counted IGF‐1R immunoreactive and non‐immunoreactive cells in the SON of young‐ad‐libitum fed (young‐Al, 6 weeks), old‐ad‐libitum fed (Old‐Al, 22 months), and old‐calorie‐restricted (Old‐CR, 22 months) female B6D2F1 mice. An automated imaging microscopy system (AIMS) was used to generate cell counts for each section of supraoptic hypothalamus. Results show that while the total number of cells in the SON of ad‐libitum fed mice does not change significantly with aging, a significant reduction in IGF‐1R immunoreactive cells does occur in ad‐libitum fed mice with aging. In contrast to this, calorie restricted mice show both a decline in the total number of cells and IGF‐1R immunoreactive cells in the SON with age, but with the decrease in the latter being notably attenuated when compared to the degree of loss seen in ad‐libitum fed mice. Thus, while CR induces greater loss in the total number of cells in the SON with age, it reduces the degree of age‐dependent loss seen in IGF‐1R expressing cells. As a result, when compared to Old‐AL mice, the SON of Old‐CR mice displays a greater proportion of IGF‐1R cells and thus possibly enhanced IGF‐1 sensitivity with aging.

Resuscitating hypothermic dogs after 2 hours of circulatory arrest below 6 degrees C.

BACKGROUND Ultraprofound hypothermia may have a place in trauma rescue and resuscitation. We describe resuscitation of dogs after asanguineous perfusion and circulatory arrest of 2 hours at 2 degrees to 4 degrees C. METHODS Nine dogs were cooled using a bypass apparatus and their circulating blood replaced with bicarbonated Hextend (Abbott, North Chicago, IL). Perfusion was continued to 2 degrees to 4 degrees C, and 60 mL of 2 mol/L KCl and 20 mL of 50% MgSO(4).7H(2)O were infused intra-arterially, and circulation was arrested for 2 hours. The dogs were then rewarmed, transfused, defibrillated, weaned from bypass, and allowed to awaken. Preoperative and postoperative biochemistry and hematology were compared. RESULTS Six dogs recovered fully. One of these dogs died of an infection 2 weeks later. Three other dogs never recovered because of technical or procedural difficulties. Biochemical and hematologic parameters were normal by 3 weeks. CONCLUSION Hypothermic blood substitution with Hextend allows resuscitation after 2 hours of ice-cold circulatory arrest in dogs.

Dnmt1, Dnmt3a and Dnmt3b cooperate in photoreceptor and outer plexiform layer development in the mammalian retina

Abstract Characterizing the role of epigenetic regulation in the mammalian retina is critical for understanding fundamental mechanisms of retinal development and disease. DNA methylation, an epigenetic modifier of genomic DNA, plays an important role in modulating networks of tissue and cell‐specific gene expression. However, the impact of DNA methylation on retinal development and homeostasis of retinal neurons remains unclear. Here, we have created a tissue‐specific DNA methyltransferase (Dnmt) triple mutant mouse in an effort to characterize the impact of DNA methylation on retinal development and homeostasis. An Rx‐Cre transgene was used to drive targeted mutation of all three murine Dnmt genes in the mouse retina encoding major DNA methylation enzymes DNMT1, DNMT3A and DNMT3B. The triple mutant mice represent a hypomorph model since Dnmt1 catalytic activity was still present and excision of Dnmt3a and Dnmt3b had only about 90% efficiency. Mutation of all three Dnmts resulted in global genomic hypomethylation and dramatic reorganization of the photoreceptor and synaptic layers within retina. Transcriptome and proteomic analyses demonstrated enrichment of dysregulated phototransduction and synaptic genes. The 5 mC signal in triple mutant retina was confined to the central heterochromatin but reduced in the peripheral heterochromatin region of photoreceptor nuclei. In addition, we found a reduction of the 5 mC signal in ganglion cell nuclei. Collectively, this data suggests cooperation of all three Dnmts in the formation and homeostasis of photoreceptors and other retinal neurons within the mammalian retina, and highlight the relevance of epigenetic regulation to sensory retinal disorders and vision loss. Graphical abstract Figure. No caption available. HighlightsS‐cone photoreceptors are more sensitive to loss of Dnmts than M‐cone photoreceptors.Modulation of Dnmt1, Dnmt3a and Dnmt3b levels in mammalian retina impacts synaptic proteins present in OPL and IPL.Dnmt1, Dnmt3a, Dnmt3b contribute to photoreceptor terminal differentiation, not to photoreceptor cell fate determination.Disruption of Dnmt1, Dnmt3a and Dbnmt3b reduces the 5 mC signal in ONL and GCL.

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.

Insulin-like growth factor-1 receptor immunoreactive cells are selectively maintained in the paraventricular hypothalamus of calorically restricted mice

The mammalian lifespan is dramatically extended by both caloric restriction (CR) and insulin‐like growth factor‐1 (IGF‐1) suppression. Both interventions involve neuroendocrine alterations directed by the hypothalamus. Yet, it remains unclear whether CR exerts its affects by altering central IGF‐1 sensitivity. With this question in mind, we investigated the influence of CR and normal aging on hypothalamic IGF‐1 sensitivity, by measuring the changes in IGF‐1 receptor (IGF‐1R) populations. Taking IGF‐1 receptor (IGF‐1R) immunoreactivity as an index of sensitivity to IGF‐1, we counted IGF‐1R immunoreactive and non‐immunoreactive cells in the paraventricular nucleus (PVN) of Young‐ad libitum fed (Young‐Al, 6 weeks old), Old‐ad libitum fed (Old‐Al, 22 months old), and old calorically restricted (Old‐CR, 22 months old) female B6D2F1 mice. An automated imaging microscopy system (AIMS) was used to generate cell counts for each cross‐section of PVN hypothalamus. Ad libitum fed mice show a 37% reduction in IGF‐1R immunoreactive cells and a 12% reduction in the total cell population of the PVN with aging. In comparison, caloric‐restricted mice show a 33% reduction in IGF‐1R immunoreactive cells and a notable 24% decrease in the total cell population with aging. This selective maintenance of IGF‐1R expressing cells coupled with the simultaneous loss of non‐immunoreactive cells, results in a higher percentage of IGF‐1R immunoreactive cells in the PVNs of CR mice. Thus, the decline in the percentage of IGF‐1 sensitive cells in the PVN with age is attenuated by CR.