High-resolution whole genome tiling path array CGH analysis of CD34+ cells from patients with low-risk myelodysplastic syndromes reveals cryptic copy number alterations and predicts overall and leukemia-free survival.

Myelodysplastic syndromes (MDSs) pose an important diagnostic and treatment challenge because of the genetic heterogeneity and poorly understood biology of the disease. To investigate initiating genomic alterations and the potential prognostic significance of cryptic genomic changes in low-risk MDS, we performed whole genome tiling path array comparative genomic hybridization (aCGH) on CD34(+) cells from 44 patients with an International Prognostic Scoring System score less than or equal to 1.0. Clonal copy number differences were detected in cells from 36 of 44 patients. In contrast, cells from only 16 of the 44 patients displayed karyotypic abnormalities. Although most patients had normal karyotype, aCGH identified 21 recurring copy number alterations. Examples of frequent cryptic alterations included gains at 11q24.2-qter, 17q11.2, and 17q12 and losses at 2q33.1-q33.2, 5q13.1-q13.2, and 10q21.3. Maintenance of genomic integrity defined as less than 3 Mb total disruption of the genome correlated with better overall survival (P = .002) and was less frequently associated with transformation to acute myeloid leukemia (P = .033). This study suggests a potential role for the use of aCGH in the clinical workup of MDS patients.

Feeder-free and serum-free in vitro assay for measuring the effect of drugs on acute and chronic myeloid leukemia stem/progenitor cells.

Research on chronic and acute myeloid leukemia (CML/AML) is focused on the development of novel therapeutic strategies to eliminate leukemic stem/progenitor cells that are responsible for drug resistance and disease relapse. Methods to culture hematopoietic stem/progenitor cells (HSPCs) from blood or bone marrow samples are indispensable for investigating disease pathogenesis and delineating drug responses in individual patients. A key challenge in this area is that primary leukemic cells grow poorly in culture or rapidly differentiate and lose their hematopoietic potential. Access to patient samples can also be limiting or cell numbers too low to enable large-scale assays and/or to obtain reproducible quantitative data. Here we describe a feeder cell-free and serum-free liquid culture system for the expansion of CD34+ HSPCs from CML/AML samples and healthy control tissues. Following 7 or 14 days of culture, CD34+ cells are expanded 30- to 65-fold or 400- to 800-fold, yielding a purity of ∼80% and ∼60% CD34+ cells, respectively. This system was adapted to a 96-well format to measure the sensitivity of leukemic and normal HSPCs to cytotoxic drugs after only 7 days. The assay requires only 103 cells per well to determine drug IC50 values and can be performed with uncultured and culture-expanded cells. Importantly, resulting IC50 values strongly correlate with those obtained in the classic colony-forming unit (CFU) assay. Compared with the CFU assay, this novel 96-well liquid-based assay designed specifically for leukemic and normal HSPCs is faster and simpler, with more flexible readout methods for selecting candidates for further drug development.

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.

Isolation of subsets of immune cells.

Subsets of immune cells can be isolated before analysis by the enzyme-linked immunospot (ELISPOT) assay with various cell separation techniques. This chapter describes techniques to select desired cells or deplete unwanted cells by crosslinking cells to dense or magnetic particles for subsequent separation. The RosetteSep method can be used to isolate specific cell types directly from human whole blood, using the red blood cells (RBCs) present in the sample as dense particles. Unwanted cells are crosslinked to multiple RBCs, forming "rosettes." The rosettes, free RBCs, and granulocytes pellet when the sample is centrifuged over a buoyant density medium. The unlabeled, desired cells are simply collected from the interface between the plasma and the buoyant density medium. The SpinSep method for isolation of mouse spleen or bone marrow cells is similar to RosetteSep, except that the unwanted cells are bound to dense particles rather than RBCs. The EasySep immunomagnetic system can be used with cell suspensions from a variety of species. Cells are crosslinked to nanometer-sized paramagnetic particles. Magnetically labeled cells are separated from unlabeled cells by placing the sample in a high gradient magnetic field. Both the labeled and the unlabeled fractions can be recovered for further use.

Identification and isolation of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are defined by their ability to repopulate all of the hematopoietic lineages in vivo and sustain the production of these cells for the life span of the individual. In the absence of reliable direct markers for HSCs, their identification and enumeration depends on functional long-term, multilineage, in vivo repopulation assays. The extremely low frequency of HSCs in any tissue and the absence of a specific HSC phenotype have made their purification and characterization a highly challenging goal. HSCs and primitive hematopoietic cells can be distinguished from mature blood cells by their lack of lineage-specific markers and presence of certain other cell-surface antigens, such as CD133 (for human cells) and c-kit and Sca-1 (for murine cells). Functional analyses of purified subpopulations of primitive hematopoietic cells have led to the development of several procedures for isolating cell populations that are highly enriched in cells with in vivo stem cell activity. Simplified methods for obtaining these cells at high yield have been important to the practical exploitation of such advances. This article reviews recent progress in identifying human and mouse HSCs and current techniques for their purification.