Terry Thomas

Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.

Advancement in our understanding of the biology of adult stem cells and their therapeutic potential relies heavily on meaningful functional assays that can identify and measure stem cell activity in vivo and in vitro. In the mammalian nervous system, neural stem cells (NSCs) are often studied using a culture system referred to as the neurosphere assay. We previously challenged a central tenet of this assay, that all neurospheres are derived from a NSC, and provided evidence that it overestimates NSC frequency, rendering it inappropriate for quantitation of NSC frequency in relation to NSC regulation. Here we report the development and validation of the neural colony‐forming cell assay (NCFCA), which discriminates stem from progenitor cells on the basis of their proliferative potential. We anticipate that the NCFCA will provide additional clarity in discerning the regulation of NSCs, thereby facilitating further advances in the promising application of NSCs for therapeutic use.

Retroviral marking of acute myelogenous leukemia progenitors that initiate long-term culture and growth in immunodeficient mice

Rare primitive progenitors among the malignant cells from most patients with AML include AML long-term culture-initiating cells (AML LTC-IC) and NOD/SCID mouse leukemia-initiating cells (NOD/SL-IC). To evaluate the feasibility of genetic modification of these progenitors for gene marking and/ or gene therapy strategies, cells from patients with newly-diagnosed AML were cocultured with retroviral producer cells and then placed in colony (AML-CFC) assays, LTC, and injected intravenously into NOD/SCID mice. Southern blotting demonstrated transfer of the neo(r) gene to 30% to 80% of leukemic blasts when cells were cultured for 48 hours in the presence of IL-3 and steel factor (SF) prior to 48-hour coculture with viral producers. Three of six retrovirally-infected AML samples showed both engraftment in NOD/SCID mice and the presence of the neo(r) transgene in mouse tissues 8-15 weeks after injection of transduced cells. Thirteen weeks after injection of one of these samples, >80% of cells from mouse bone marrow were the progeny of two retrovirally-transduced AML progenitors. Four of the remaining five samples showed markedly reduced ability to engraft in mice after retroviral infection. Subsequent experiments demonstrated that the loss of engraftment potential took place within 24 hours of culture initiation in the absence of retroviral producers and regardless of the cytokines present. Interestingly, the majority of AML-CFC or AML LTC-IC survived the 24-hour culture period. A retroviral vector containing the murine cell surface marker heat stable antigen (HSA), which allows purification of transduced cells on immunomagnetic columns, was used to obtain an enriched population of gene-modified AML cells following an infection protocol that eliminated the 48 hours of prestimulation in IL-3 and SF and reduced coculture with viral producers to 10-36 hours. These modifications failed to improve engraftment of the infected cells. In addition, in these experiments more than 10 hours of cocultivation with viral producer cells was necessary to achieve gene transfer and expression in AML LTC-IC. These data demonstrate that although retroviral-mediated gene transfer can be achieved to AML progenitors, including NOD/SL-IC, improved culture conditions will be required before substantial numbers of such transduced primitive progenitors can be obtained. In addition, the difference in the ability of AML LTC-IC and NOD/SL-IC to survive ex vivo suggests that these assays may detect different populations of cells or that changes are induced in vitro in primitive cells which can only be detected in the mouse assay.

Flow Cytometry and Immunoselection of Human Stem Cells

Human hematopoietic stem cells (HSCs) can be obtained from a variety of hematopoietic tissues, including bone marrow, blood, cord blood, and fetal liver. Various techniques have been used to fractionate hematopoietic cell populations based on differences in size and density, expression of cell-surface antigens, differential dye uptake, and sensitivity to cytotoxic drugs. The very low frequency of HSCs in hematopoietic tissues presents an enormous challenge to purification strategies aimed at isolation of sufficient cells of suitablpurity for further study. The most effective approaches invariably involve several cell-separation steps which differ in capacity and degree of selectivity.

A defined, feeder-independent medium for human embryonic stem cell culture

Human embryonic stem cells (hESC) are derived from the epiblast tissue of the inner cell mass and are capable of giving rise to cells from all three germ layers - ectoderm, mesoderm, and endoderm. Although the basic techniques to create and culture hESC are well established, limitations remain in many of the procedures used for their culture. In particular, most existing hESC lines have been cultured with components derived from animal serum and using mouse embryonic fibroblast feeder layers. However, the use of serum or feeder layers induces variability in these culture systems, and limits their application in the clinical setting. Accordingly, there is considerable interest in the development of serum-free and feeder-independent hESC culture systems. mTeSR™1 is a fully-defined, serum-free medium for the derivation and maintenance of hESCs cultured on BD Matrigel™ hESC-qualified Matrix(Ludwig et al. Nat Methods 3(8):637-46). mTeSR™1 has been demonstrated to support the undifferentiated culture of multiple hESC lines derived by independent sources, with examples including H1, H9 CA1, and SA121. Cells grown for multiple passages in mTeSR™1 maintain characteristic undifferentiated colony morphology, and FACS analysis confirms high expression of pluripotency-associated markers (Oct4, SSEA-3, SSEA4, TRA-1-60, and TRA-1-81), and the absence of expression of the differentiation marker SSEA-1. RT-PCR analysis of cells cultured in mTeSR™1 also validate the continued expression of Oct4 and Nanog and the lack of expression of multiple differentiation markers. Furthermore, hESC cultures maintained in mTeSR™1 retain a high proliferative capacity and normal karyotype after extended culture. Finally, the teratoma formation assay verified the preserved potential of these cells to subsequently differentiate into tissues derived from all three germ layers. Together these results establish mTeSR™1 as an optimal, standardized medium for researchers culturing hESC.

Characterisation of dendritic cells generated using stemspan™ serum-free medium

Abstract Mature dendritic cells (DC) are often generated in serum-replete medium. One of the disadvantages of this system is the batch-to-batch variation in the cytokine content of serum-replete medium and the consequent variation in the functional properties of the DC generated. A serum-free medium, in which cytokine content is controlled is therefore preferable. Here we describe the use of StemSpan™ Serum-Free Medium for the generation of DC from a variety of sources. Lineage negative bone marrow cells (30-50% CD34+) were enriched using the StemSep™ immuno-magnetic negative selection procedure and cultured in StemSpan™ SFEM supplemented with GM-CSF and FLT3-ligand. After 14 days in culture, the cells lost expression of CD34, developed typical DC morphology, increased expression of costimulatory molecules and gained the ability to stimulate an MLR. In addition, these CD34-derived DC were able to present peptide to CD8+ T cells. Peripheral blood monocytes (CD14+) isolated using StemSep™ were cultured for 7days in StemSpan™ SFEM supplemented with GM-CSF (100ng/ml) and IL4 (20ng/ml). These cells developed typical DC morphology, lost expression of CD14, increased expression of CD86, CD40, CD1a and HLA-DR and increased their ability to stimulate an MLR. Peripheral blood DC (defined as CD4+ HLA− DR+lin−) were enriched using StemSep™ and cultured in StemSpan™ SFEM with GM-CSF (100ng/ml) and TNFa (10ng/ml). After 3 days, these cells developed a typical DC morphology, increased expression of costimulatory molecules and increased ability to stimulate an MLR. In summary, StemSpan™ SFEM, when supplemented with the appropriate cytokines, can be used to generate functionally competent DC from blood and bone marrow precursors.

Abstract 511: Unbiased enrichment of circulating tumor cells directly from whole blood

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Enrichment of rare circulating tumor cells (CTC) in peripheral blood is required prior to most analytic procedures. The ideal enrichment method would be rapid ( 0.05) or pooled and compared to the pooled RosetteSep™ controls (Least Squares Fit; Prob F>0.05). Over all magnet separation conditions and samples, the mean log depletion of CD45+ cells was 2.9±0.4, the mean recovery of CAMA cells was 42±23%, and the residual RBC contamination was ∼9000 RBC/mL of start sample. Rare cells were enriched in 25 minutes using EasySep™ Direct and in less than 40 minutes using RosetteSep™ with SepMate™. CTC can be enriched directly from whole blood with either EasySep™ Direct or RosetteSep™. Since enriched cells are unlabeled there is nothing to interfere with subsequent further enrichment, culture, or evaluation. Citation Format: Carrie E. Peters, Danay Maestre-Battle, Steven M. Woodside, Terry E. Thomas, Allen C. Eaves. Unbiased enrichment of circulating tumor cells directly from whole blood. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 511.

Abstract 1451: Microfluidic-based unbiased enrichment (negative selection) of circulating non-hematopoietic tumor cells directly from whole blood without centrifugation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The enumeration and analysis of circulating non-hematopoietic tumor cells (CTCs) is of increasing interest for monitoring disease progression or response to treatment, specifically as a companion diagnostic for new anti-cancer drugs, and for research into the mechanisms of disease progression and metastases. Ideally, CTCs would be enriched from very small samples, with minimal handling, high recovery, and no requirement for the expression of specific surface markers. Two technologies have been combined to meet these requirements. Hematopoietic white blood cells (WBCs) in whole blood were first cross-linked to magnetic particles using EasySep™ anti-CD45 TAC. The sample was diluted and placed in a magnet for 30 min.; an outlet in the bottom of the sample tube was then opened and the sample flowed by gravity into a microfluidic chamber containing a high-precision micro-slit membrane. Red blood cells (RBCs) flowed through the microfluidic chamber, while larger cells such as CTCs were retained in the chamber. The cells in the chamber were washed with PBS and then identified by staining with Hoechst [nuclear], anti-cytokeratin antibodies [epithelial cells], and anti-CD45 antibodies [hematopoietic cells]. CTCs were defined as Hoechst+, cytokeratin+ and CD45-. The recovery of MCF-7 breast adenocarcinoma cells spiked into normal whole blood, at 10, 30, 50, or 100 cells / 2 mL blood was 95 ± 23% (7 ± 1, n=3 for 10 cells; 24 ± 3, n=3 for 30 cells; 57 ± 3, n=3 for 50 cells; 113 ± 27, n=3 for 100 cells) and the log depletion of WBCs exceeded 2.3. The recovery of H1975 lung adenocarcinoma cells spiked into normal whole blood, at 10, 30, 50, or 100 cells / 2 mL blood was 93 ± 8% (9 ± 1, n=3 for 10 cells; 28 ± 3, n=3 for 30 cells; 48 ± 6, n=3 for 50 cells; 94 ± 2, n=3 for 100 cells), and the log depletion of WBCs exceeded 2.14. 13 patient samples [10 NSCLC and 3 CRC] were processed with this method and CTCs were detected in every sample. The number of CTC detected from 2 mL of blood ranged from 1 to 51. WBC log depletion ranged from 2.01 to 2.79. No RBCs were observed on the membrane of the microfluidic chamber. The entire process requires ∼ 60 minutes and could easily be automated. RBC depletion is essentially complete without the use of centrifugation or chemicals which may be deleterious to CTCs. The minimal sample handling permits high recovery of desired cells, allowing the detection of CTCs in much smaller samples than are currently used for clinical evaluation. CTCs are enriched without bias as to their surface antigen expression, and are not labeled with antibodies prior to detection. CTCs can be stained and visualized directly on the microfluidic chip. This unbiased enrichment approach could be used to assess the mutation status of CTC in real time. Citation Format: Carrie E. Peters, Hamizah Ahmad, Drew Kellerman, Bhuvanendran Nair Gourikutty Sajay, Chang Chia-Pin, Wong Chee Chung, Abdur Rub Abdur Rahman, Karina L. McQueen, Terry E. Thomas. Microfluidic-based unbiased enrichment (negative selection) of circulating non-hematopoietic tumor cells directly from whole blood without centrifugation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1451. doi:10.1158/1538-7445.AM2013-1451

Abstract 2375: Rapid unbiased enrichment (negative selection) of circulating non-hematopoietic tumor cells directly from whole blood

There is increasing interest in analyzing circulating non-hematopoietic tumor cells (CTC) in peripheral blood to evaluate disease progression or response to treatment; however, CTC enrichment is required prior to most analytic procedures. The ideal enrichment method would be rapid, permit a high recovery of viable CTC, and would be independent of the expression of specific epithelial cell surface markers, since CTCs in the peripheral blood may be undergoing EMT (epithelial mesenchymal transition) and may not express epithelial markers. RosetteSep™ CD45 depletion of hematopoietic cells directly from whole blood meets these criteria. However, RosetteSep™ enrichment of CTC involves density gradient centrifugation, which entails careful layering of the sample over the density gradient medium and careful pipetting to remove the enriched cells after centrifugation. Centrifugation must be performed with the brake off to avoid disturbing the enriched cell layer, further lengthening the process. SepMate™, a centrifugation tube with a specialized insert, was developed to minimize mixing of the sample with the density gradient medium, thus allowing rapid layering of the sample on the density gradient medium and easy pouring off of the enriched cells after centrifugation. We compared CTC enrichment using RosetteSep™ and the standard tubes and protocol with RosetteSep™ using SepMate™ tubes and reduced cocktail incubation and spin times on 5 donor whole blood samples seeded with ∼1% CAMA cells. Purity of viable (PI negative) CTC obtained with SepMate™ with RosetteSep™ was 85 ± 7%; purity of CTC obtained with RosetteSep™ alone was 91 ± 5% (no significant difference, paired t test, p=0.050). Purity of CTC obtained with density gradient centrifugation only (no RosetteSep™), either with or without SepMate™, was 4 ± 2%. There was no significant difference in the recovery of enriched CTC under any of the conditions tested (RosetteSep™ ± SepMate™, SepMate™ alone, density gradient separation alone, Tukey-Kramer test, p>0.05). CTC enrichment was accomplished in Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2375. doi:1538-7445.AM2012-2375