James L. Sherley

Asymmetric Cell Kinetics Genes: The Key to Expansion of Adult Stem Cells in Culture

A singular challenge in stem cell research today is the expansion and propagation of functional adult stem cells. Unlike embryonic stem cells, which are immortal in culture, adult stem cells are notorious for the difficulty encountered when attempts are made to expand them in culture. One overlooked reason for this difficulty may be the inherent asymmetric cell kinetics of stem cells in postnatal somatic tissues. Senescence is the expected fate of a culture whose growth depends on adult stem cells that divide with asymmetric cell kinetics. Therefore, the bioengineering of strategies to expand adult stem cells in culture requires knowledge of cellular mechanisms that control asymmetric cell kinetics. The properties of several genes recently implicated to function in a cellular pathway(s) that regulates asymmetric cell kinetics are discussed. Understanding the function of these genes in asymmetric cell kinetics mechanisms may be the key that unlocks the adult stem cell expansion problem.

CXCR6, a Newly Defined Biomarker of Tissue-Specific Stem Cell Asymmetric Self-Renewal, Identifies More Aggressive Human Melanoma Cancer Stem Cells

Background A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells. Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability. In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main observations are reported. The first one is the first phenotypic evidence to support the origin of melanoma cancer stem cells (CSCs) from mutated tissue-specific stem cells; and the second one is the identification of a more aggressive subpopulation of CSCs in melanoma that are CXCR6+. Methods/Findings We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal. Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated. Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells. In addition, ABCG2+ cells produced tumors that had a 2-fold greater mass than tumors produced by unsorted cells or ABCG2- cells. CXCR6+ cells produced more aggressive tumors. CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive MCSC phenotype than cells selected on the basis of the ABCG2+ phenotype alone. Conclusions/Significance The association of a more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology. Namely, the retention of some tissue-specific stem cell attributes, like the ability to asymmetrically self-renew, impacts the natural history of human tumor development. Knowledge of this new aspect of tumor development and progression may provide new targets for cancer prevention and treatment.

Immortal DNA Strand Cosegregation Requires p53/IMPDH–Dependent Asymmetric Self-renewal Associated with Adult Stem Cells

Because they are long-lived and cycle continuously, adult stem cells (ASCs) are predicted as the most common precursor for cancers in adult mammalian tissues. Two unique attributes have been proposed to restrict the carcinogenic potential of ASCs. These are asymmetric self-renewal that limits their number and immortal DNA strand cosegregation that limits their accumulation of mutations due to DNA replication errors. Until recently, the molecular basis and regulation of these important ASC-specific functions were unknown. We developed engineered cultured cells that exhibit asymmetric self-renewal and immortal DNA strand cosegregation. These model cells were used to show that both ASC-specific functions are regulated by the p53 cancer gene. Previously, we proposed that IMP dehydrogenase (IMPDH) was an essential factor for p53-dependent asymmetric self-renewal. We now confirm this proposal and provide quantitative evidence that asymmetric self-renewal is acutely sensitive to even modest changes in IMPDH expression. These analyses reveal that immortal DNA strand cosegregation is also regulated by IMPDH and confirm the original implicit precept that immortal DNA strand cosegregation is specific to cells undergoing asymmetric self-renewal (i.e., ASCs). With IMPDH being the rate-determining enzyme for guanine ribonucleotide (rGNP) biosynthesis, its requirement implicates rGNPs as important regulators of ASC asymmetric self-renewal and immortal DNA strand cosegregation. An in silico analysis of global gene expression data from human cancer cell lines underscored the importance of p53-IMPDH-rGNP regulation for normal tissue cell kinetics, providing further support for the concept that ASCs are key targets for adult tissue carcinogenesis.

Methylation of episomal plasmids as a barrier to transient gene expression via a synthetic delivery vector.

Efficient and sustained transgene expression are desirable features for many envisioned gene therapy applications, yet synthetic vectors tested to date are rarely successful in achieving these properties. Substantial research efforts have focused on protection of plasmid DNA from nuclease attack as well as increasing nuclear transport of plasmids, resulting in significant but still limited gains. We show here that a further barrier to efficient and sustained expression exists for synthetic vectors: plasmid DNA methylation. We have investigated this barrier for transient expression of a green fluorescent protein (GFP) transgene delivered via Lipofectamine, by testing the effects of culturing C3A human hepatoblastoma cells with 5-Azacytidine (AzaC), an irreversible inhibitor of DNA methyltransferase. To control for loss of plasmids by dilution during mitosis, transfected cells were growth-arrested for 1 week and their subsequent GFP expression quantified by FACS. In the presence of AzaC, a significantly greater fraction of transfected cells remained GFP-positive and possessed higher levels of GFP production relative to AzaC-untreated cells. Additionally, we have applied a Methyl-Assisted PCR (MAP) assay to quantify a subset of methylated CpG sites in the GFP gene. When MAP was performed on plasmids isolated from transfected cells, the extent of methylation was found to be inversely related to the level of GFP expression.

CFP and YFP, but Not GFP, Provide Stable Fluorescent Marking of Rat Hepatic Adult Stem Cells

The stable expression of reporter genes in adult stem cells (ASCs) has important applications in stem cell biology. The ability to integrate a noncytotoxic, fluorescent reporter gene into the genome of ASCs with the capability to track ASCs and their progeny is particularly desirable for transplantation studies. The use of fluorescent proteins has greatly aided the investigations of protein and cell function on short-time scales. In contrast, the obtainment of stably expressing cell strains with low variability in expression for studies on longer-time scales is often problematic. We show that this difficulty is partly due to the cytotoxicity of a commonly used reporter, green fluorescent protein (GFP). To avoid GFP-specific toxicity effects during attempts to stably mark a rat hepatic ASC strain and, therefore, obtain stable, long-term fluorescent ASCs, we evaluated cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), in addition to GFP. Although we were unable to derive stable GFP-expressing strains, stable fluorescent clones (up to 140 doublings) expressing either CFP or YFP were established. When fluorescently marked ASCs were induced to produce differentiated progeny cells, stable fluorescence expression was maintained. This property is essential for studies that track fluorescently marked ASCs and their differentiated progeny in transplantation studies.

Biological principles for ex vivo adult stem cell expansion.

Adult stem cells (ASCs) are the engines that drive the renewal of adult mammalian tissues. They divide continuously, throughout life, to produce new progeny cells that undergo a robust development program of differentiation and maturation to replace older expired tissue cells. The same cell turnover program may function to provide limited repair and regeneration of adult tissues in some cases. The regenerative potential of ASCs drives the current intense interest in adapting them for applications in cell replacement therapy. However, research to explore this potential has been blunted by an unyielding biological problem. ASCs have proven highly refractory to expansion of their numbers and long-term propagation in culture. A review of reported strategies to overcome this problem reveals that many studies focus on traditional cell culture factors that may not apply to ASCs and overlook a special property of ASCs that may be universally critical for successful expansion, asymmetric cell kinetics (ACK). This property is reflected by the different kinetics fate of the two sister cells resulting from an ASC division: one cell remains an ASC and keeps the potential to divide for the entire life span of the tissue, while the other cells progeny eventually differentiates and undergoes terminal division arrest. This unique property of ASCs may prove to be the obligatory factor that must be breached by any method that will succeed in accomplishing routine expansion of ASCs of diverse tissue origin.

Molecular Cloaking of H2A.Z on Mortal DNA Chromosomes During Nonrandom Segregation

Although nonrandom sister chromatid segregation is a singular property of distributed stem cells (DSCs) that are responsible for renewing and repairing mature vertebrate tissues, both its cellular function and its molecular mechanism remain unknown. This situation persists in part because of the lack of facile methods for detecting and quantifying nonrandom segregating cells and for identifying chromosomes with immortal DNA strands, the cellular molecules that signify nonrandom segregation. During nonrandom segregation, at each mitosis, asymmetrically self‐renewing DSCs continuously cosegregate to themselves the set of chromosomes that contain immortal DNA strands, which are the oldest DNA strands. Here, we report the discovery of a molecular asymmetry between segregating sets of immortal chromosomes and opposed mortal chromosomes (i.e., containing the younger set of DNA template strands) that constitutes a new convenient biomarker for detection of cells undergoing nonrandom segregation and direct delineation of chromosomes that bear immortal DNA strands. In both cells engineered with DSC‐specific properties and ex vivo‐expanded mouse hair follicle stem cells, the histone H2A variant H2A.Z shows specific immunodetection on immortal DNA chromosomes. Cell fixation analyses indicate that H2A.Z is present on mortal chromosomes as well but is cloaked from immunodetection, and the cloaking entity is acid labile. The H2A.Z chromosomal asymmetry produced by molecular cloaking provides a first direct assay for nonrandom segregation and for chromosomes with immortal DNA strands. It also seems likely to manifest an important aspect of the underlying mechanism(s) responsible for nonrandom sister chromatid segregation in DSCs. STEM CELLS 2011;29:1620–1627

Comparison of bax, waf1, and IMP dehydrogenase regulation in response to wild‐type p53 expression under normal growth conditions

Recently, we demonstrated that downregulation of inosine‐5′‐monophosphate dehydrogenase (IMPD; IMP:NAD oxidoreductase, EC 1.2.1.14), the rate‐limiting enzyme for guanine nucleotide biosynthesis, is required for p53‐dependent growth suppression. These studies were performed with cell lines derived from immortal, nontumorigenic fibroblasts that express wild‐type p53 conditionally by virtue of a metal‐responsive promoter. Here, the p53‐dependent properties of the original “p53‐inducible” fibroblasts are presented in detail and compared to related properties of epithelial cells that also express wild‐type p53 conditionally, but by virtue of a temperature‐responsive promoter. Both types of p53‐inducible cells were designed to approximate normal physiologic relationships between the host cell and the regulated p53 protein. Together, they were used to investigate expression relationships between IMPD and other p53‐responsive genes proposed as mediators of p53‐dependent growth suppression. In both types of cells, IMPD activity, protein, and mRNA were consistently coordinately reduced in response to p53 expression. In contrast, mRNAs for waf1, bax, and mdm2 showed disparate patterns of expression, being induced in one conditional cell type, but not the other. This distinction in regulation pattern suggests that under normal growth conditions, unlike IMPD downregulation, bax and waf1 induction is not a rate‐determining event for p53‐dependent growth suppression. J. Cell. Physiol. 177:364–376, 1998.

Higher 5-hydroxymethylcytosine identifies immortal DNA strand chromosomes in asymmetrically self-renewing distributed stem cells

Significance Distributed stem cells (DSCs), which continuously divide asymmetrically to replenish mature tissue cells, adopt a special form of mitotic chromosome segregation. Chromosome segregation is nonrandom instead of random. DSCs cosegregate the set of sister chromosomes with the older of the two template DNA strands used for semiconservative DNA replication during the preceding S phase. Neither the responsible molecular mechanisms nor the cellular function of nonrandom segregation are known. Here, we report evidence that immortal strand chromosomes have a higher level of cytosine 5-hydroxymethylation than mortal chromosomes, which contain the younger DNA template strands. We propose that asymmetric chromosomal 5-hydroxymethylation is a key element of a cellular mechanism by which DSCs distinguish older DNA template strands from younger ones. Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs “know” the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency.

A Resource for Discovering Specific and Universal Biomarkers for Distributed Stem Cells

Specific and universal biomarkers for distributed stem cells (DSCs) have been elusive. A major barrier to discovery of such ideal DSC biomarkers is difficulty in obtaining DSCs in sufficient quantity and purity. To solve this problem, we used cell lines genetically engineered for conditional asymmetric self-renewal, the defining DSC property. In gene microarray analyses, we identified 85 genes whose expression is tightly asymmetric self-renewal associated (ASRA). The ASRA gene signature prescribed DSCs to undergo asymmetric self-renewal to a greater extent than committed progenitor cells, embryonic stem cells, or induced pluripotent stem cells. This delineation has several significant implications. These include: 1) providing experimental evidence that DSCs in vivo undergo asymmetric self-renewal as individual cells; 2) providing an explanation why earlier attempts to define a common gene expression signature for DSCs were unsuccessful; and 3) predicting that some ASRA proteins may be ideal biomarkers for DSCs. Indeed, two ASRA proteins, CXCR6 and BTG2, and two other related self-renewal pattern associated (SRPA) proteins identified in this gene resource, LGR5 and H2A.Z, display unique asymmetric patterns of expression that have a high potential for universal and specific DSC identification.