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ABSTRACTHuman induced pluripotent stem cells (iPSCs) are an invaluable endless cell source for generating various therapeutic cells and tissues. However,their differentiation into specific cell lineages,such as definitive endoderm (DE) and pancreatic progenitor (PP),often suffers from poor reproducibility,due partially to their pluripotency. In this work,we investigated the impact of iPSC confluency during cell self?renewal and seeding density on cell metabolic activity,glycolysis to oxidative phosphorylation shift,and differentiation potential toward DE and PP lineages. Our findings demonstrated that cell seeding strategy influences cellular metabolic activity and the robustness of iPSC differentiation. iPSCs maintained at higher seeding density exhibited lower initial oxygen consumption rate (OCR) and metabolic activity. There is an optimal seeding density to ensure sufficient oxygen consumption during differentiation and to yield high expression of SOX17 in the DE lineage and high PDX1/NKX6.1 dual?positive cells in PPs. Interestingly,we found that cell confluency at the time of harvest has less impact on the efficacy of pancreatic lineage formation or metabolic activity. This study sheds light on the interplay between metabolic activity and iPSC lineage specification,offering new insights into the robustness of iPSC self?renewal and differentiation for creating human tissues. Graphical Abstract and Lay SummaryHuman induced pluripotent stem cell (iPSC) differentiation into specific cell lineages often shows poor reproducibility due to cell pluripotency. This study demonstrated impact of iPSC seeding strategy on metabolic activity and differentiation potential,offering new insights into the robustness of iPSC self?renewal and differentiation for creating human tissues. View Publication

The effector potential of NK cells is counterbalanced by their sensitivity to inhibition by self" MHC class I molecules in a process called "education." In humans� View Publication

Plasmacytoma growth factor (PCT-GF),a putative macrophage-derived lymphokine essential for the in vitro viability and proliferation of early generation plasmacytomas,was purified from conditioned medium of the murine macrophage cell line P388D1. The purification of PCT-GF was accomplished by a batch concentration on trimethylsilyl-controlled pore glass beads,followed by: gel filtration chromatography; hydrophobic interaction HPLC; and reverse-phase HPLC. SDS-PAGE analysis of the purified PCT-GF revealed a single band of Mr 23,000. The amino terminal sequence of PCT-GF was established as NH2-Pro-Thr-Ser-Gln-Val-Arg-Arg-Gly-Asp-Phe-Thr-Glu-Asp-Thr-Thr-Pro-Asn- Arg-Pro-Val-Tyr-Thr. No significant homology was found between this sequence and proteins in the National Biomedical Research Foundation database,suggesting that PCT-GF is a new lymphokine unrelated to previously described growth and differentiation factors. View Publication

The cell line described here was established for a 50-year-old male patient with rapidly progressive non-Hodgkin's lymphoma whose marrow was diffusely infiltrated with large granular lymphocytes (LGL). Immunophenotyping of marrow blasts and peripheral lymphocytes was positive for CD56,CD2 and CD7,and negative for CD3. Cytotoxicity of peripheral blood mononuclear cells at an effector: target (E:T) cell ratio of 50:1 was 79% against K562 cells and 48% against Daudi cells. To establish the line,cells from the peripheral blood were placed into enriched alpha medium containing 12.5% fetal calf serum,12.5% horse serum,10(-4) M beta-mercaptoethanol and 10(-6) M hydrocortisone. Growth of the line (termed NK-92) is dependent on the presence of recombinant IL-2 and a dose as low as 10 U/ml is sufficient to maintain proliferation. Conversely,cells die within 72 h when deprived of IL-2; IL-7 and IL-12 do not maintain long-term growth,although IL-7 induces short-term proliferation measured by 3H-thymidine incorporation. None of the other cytokines tested (IL-1 alpha,IL-6,TNF-alpha,IFN-alpha,IFN-gamma) supported growth of NK-92 cells which have the following characteristics: surface marker positive for CD2,CD7,CD11a,CD28,CD45,CD54,CD56bright; surface marker negative for CD1,CD3,CD4,CD5,CD8,CD10,CD14,CD16,CD19,CD20,CD23,CD34,HLA-DR. DNA analysis showed germline configuration for T-cell receptor beta and gamma genes. CD25 (p55 IL-2 receptor) is expressed on about 50% of all cells when tested at 100 U/ml of IL-2 and its expression correlates inversely with the IL-2 concentration. The p75 IL-2 receptor is expressed on about half of the cells at low density irrespective of the IL-2 concentration. NK-92 cells kill both K562 and Daudi cells very effectively in a 4 h51-chromium release assay (84 and 86% respectively,at an E:T cell ratio of 5:1). The cell line described here thus displays characteristics of activated NK-cells and could be a valuable tool to study their biology. View Publication

Luminal and myoepithelial cells have been separated from normal adult human breast epithelium using fluorescence activated cell sorting. Their isolation was based on the exclusive expression of two surface antigens,epithelial membrane antigen (EMA) and the common acute lymphoblastic leukaemia antigen (CALLA/CD10/neutral endopeptidase 24.11). Sorted luminal and myoepithelial cells displayed distinctively different morphologies when maintained in monolayer culture,differences which were enhanced by the addition of hydrocortisone,insulin and cholera toxin to the culture medium. The EMA-positive cells formed an attenuated monolayer with indistinct cell boundaries while CALLA-positive cells,by contrast,formed tightly packed arrays of refractile cells. The distribution of the cell type-specific markers cytokeratin 18 (luminal cells) and smooth muscle alpha-actin (myoepithelial cells) indicated that the sorted populations were approximately 98% pure. However,a significant minority (approximately 15%) of sorted luminal cells consistently expressed the basal-cell marker cytokeratin 14 in culture. A marked difference was noted in the proliferative behaviour of the two types of sorted cells,with myoepithelial cells dividing rapidly in response to the humoural additives,in contrast to the luminal cells which proliferated slowly. Both types of sorted cells could be cloned in the presence of feeder layers of mouse fibroblasts. Clones of luminal and myoepithelial cells were also distinctive; all spread" luminal clones were similar in appearance to each other� View Publication

Primary systemic amyloidosis (AL) is a rare monoclonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin (Ig) light chains (LC) in vital organs throughout the body. To our knowledge,no cell lines have ever been established from AL patients. Here we describe the establishment of the ALMC-1 and ALMC-2 cell lines from an AL patient. Both cell lines exhibit a PC phenotype and display cytokine-dependent growth. Using a comprehensive genetic approach,we established the genetic relationship between the cell lines and the primary patient cells,and we were also able to identify new genetic changes accompanying tumor progression that may explain the natural history of this patient's disease. Importantly,we demonstrate that free lambda LC secreted by both cell lines contained a beta structure and formed amyloid fibrils. Despite absolute Ig LC variable gene sequence identity,the proteins show differences in amyloid formation kinetics that are abolished by the presence of Na(2)SO(4). The formation of amyloid fibrils from these naturally secreting human LC cell lines is unprecedented. Moreover,these cell lines will provide an invaluable tool to better understand AL,from the combined perspectives of amyloidogenic protein structure and amyloid formation,genetics,and cell biology. View Publication

We have previously demonstrated that lineage negative cells (Lin(neg)) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone,muscle,endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin(neg) UCB cells to differentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin,neurofilament,A2B5 and Sox2) are expressed in Lin(neg) cells cultured in FGF4,SCF,Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendrocyte markers CNPase,GalC,Olig2 and the late-stage marker MOSP are observed,as is the Schwann cell marker PMP22. In summary,Lin(neg) UCB cells,when appropriately cultured,are able to exhibit characteristics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schwann cells and express proteins associated with myelin production after in vitro differentiation. View Publication

Tumors may be initiated and maintained by a cellular subcomponent that displays stem cell properties. We have used the expression of aldehyde dehydrogenase as assessed by the ALDEFLUOR assay to isolate and characterize cancer stem cell (CSC) populations in 33 cell lines derived from normal and malignant mammary tissue. Twenty-three of the 33 cell lines contained an ALDEFLUOR-positive population that displayed stem cell properties in vitro and in NOD/SCID xenografts. Gene expression profiling identified a 413-gene CSC profile that included genes known to play a role in stem cell function,as well as genes such as CXCR1/IL-8RA not previously known to play such a role. Recombinant interleukin-8 (IL-8) increased mammosphere formation and the ALDEFLUOR-positive population in breast cancer cell lines. Finally,we show that ALDEFLUOR-positive cells are responsible for mediating metastasis. These studies confirm the hierarchical organization of immortalized cell lines,establish techniques that can facilitate the characterization of regulatory pathways of CSCs,and identify potential stem cell markers and therapeutic targets. View Publication

Cripto is a developmental oncoprotein that signals via mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK),phosphatidylinositol 3-kinase (PI3K)/Akt and Smad2/3 pathways. However,the molecular basis for Cripto coupling to these pathways during embryogenesis and tumorigenesis is not fully understood. In this regard,we recently demonstrated that Cripto forms a cell surface complex with the HSP70 family member glucose-regulated protein-78 (GRP78). Here,we provide novel functional evidence demonstrating that cell surface GRP78 is a necessary mediator of Cripto signaling in human tumor,mammary epithelial and embryonic stem cells. We show that targeted disruption of the cell surface Cripto/GRP78 complex using shRNAs or GRP78 immunoneutralization precludes Cripto activation of MAPK/PI3K pathways and modulation of activin-A,activin-B,Nodal and transforming growth factor-beta1 signaling. We further demonstrate that blockade of Cripto binding to cell surface GRP78 prevents Cripto from increasing cellular proliferation,downregulating E-Cadherin,decreasing cell adhesion and promoting pro-proliferative responses to activin-A and Nodal. Thus,disrupting the Cripto/GRP78 binding interface blocks oncogenic Cripto signaling and may have important therapeutic value in the treatment of cancer. View Publication

BACKGROUND: The presence of chromosomal abnormalities could have a negative impact for human embryonic stem cell (hESC) applications both in regenerative medicine and in research. A biomarker that allows the identification of chromosomal abnormalities induced in hESC in culture before they take over the culture would represent an important tool for defining optimal culture conditions for hESC. Here we investigate the expression of CD30,reported to be a biomarker of hESCs with abnormal karyotype,in undifferentiated and spontaneously differentiated hESC.backslashnbackslashnMETHODS AND RESULTS: hESC were derived and cultured on mouse fibroblasts in KO-SR containing medium (serum free media) and passaged mechanically. Our results based on analysis at mRNA (RT-PCR) and protein (fluorescence-activated cell sorting and immunocytochemistry) level show that CD30 is expressed in undifferentiated hESC,even at very early passages,without any correlation with the presence of chromosomal anomalies. We also show that the expression of CD30 is rapidly lost during early spontaneous differentiation of hESC.backslashnbackslashnCONCLUSION: We conclude that CD30 expression in hESC cultures is probably a consequence of culture conditions,and that KO-SR may play a role. In addition,the expression of so-called 'stemness' markers does not change in undifferentiated hESC during long-term culture or when cells acquire chromosomal abnormalities. View Publication

Chemokine receptor CX3CR1(+) dendritic cells (DCs) have been suggested to sample intestinal antigens by extending transepithelial dendrites into the gut lumen. Other studies identified CD103(+) DCs in the mucosa,which,through their ability to synthesize retinoic acid (RA),appear to be capable of generating typical signatures of intestinal adaptive immune responses. We report that CD103 and CX3CR1 phenotypically and functionally characterize distinct subsets of lamina propria cells. In contrast to CD103(+) DC,CX3CR1(+) cells represent a nonmigratory gut-resident population with slow turnover rates and poor responses to FLT-3L and granulocyte/macrophage colony-stimulating factor. Direct visualization of cells in lymph vessels and flow cytometry of mouse intestinal lymph revealed that CD103(+) DCs,but not CX3CR1-expressing cells,migrate into the gut draining mesenteric lymph nodes (LNs) under steady-state and inflammatory conditions. Moreover,CX3CR1(+) cells displayed poor T cell stimulatory capacity in vitro and in vivo after direct injection of cells into intestinal lymphatics and appeared to be less efficient at generating RA compared with CD103(+) DC. These findings indicate that selectively CD103(+) DCs serve classical DC functions and initiate adaptive immune responses in local LNs,whereas CX3CR1(+) populations might modulate immune responses directly in the mucosa and serve as first line barrier against invading enteropathogens. View Publication

BACKGROUND: Over-expression of aldehyde dehydrogenase and other stem cell markers is characteristic of cells with tumorigenic potential in NOD/SCID mice. Most of these studies have focused on metastatic cells in bone marrow and on solid tumors. There are no studies on correlation of marker expression with ALDH1 expression in cells from human peripheral blood apheresis (HPC-A) samples. METHODS: HPC-A samples from 44 patients were incubated with Aldefluor with or without the presence of aldehyde dehydrogenase inhibitor DEAB. Cells with high aldehyde dehydrogenase expression (ALDH1(bright)) were analyzed for stem/progenitor markers CD34,CD90,CD117,and CD133. Electronic volume measured by Coulter principal in a Quanta flow analyzer was correlated with ALDH1 and marker expression. RESULTS: In ALDH1(bright)/SSC(low) cells,0.13% of the cells had CD34(+) expression and three distinct populations were seen. Expression of CD90 was dim and the frequency of ALDH1(bright)/SSC(low)/CD90(dim) cells amongst the nonlineage depleted samples was 0.04%. CD117(dim-bright) expression was seen in 0.17% of the samples. Three distinct populations of cells with CD133 expression were seen in ALDH1(bright)/SSC(low) nonlineage depleted cells with a frequency of 0.28%. The ALDH1(bright)/CD90(dim) cells had the smallest mean electronic volume of 264.9 microm(3) when compared with cells with CD34(bright) expression (270.2 microm(3)) and ALDH1(dim)/CD90(dim) cells (223 microm(3)). CONCLUSIONS: ALDH1(bright)/SSC(low) cells show heterogeneity in expression of the four stem cell markers studied. The CD90 cells in both the ALDH1(bright) and ALDH1(dim) populations had the smallest mean electronic volume when compared with similar cells with CD117 expression. View Publication