Mardiyyah/TAPT_data_V2_split · Datasets at Hugging Face

PMCID stringclasses 24 values	Title stringclasses 24 values	Sentences stringlengths 2 40.7k
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	E) The differentiation status of EB is detected by RT-PCR using different germ layer cell markers.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Selected endoderm markers AFP, FoxA2; mesoderm markers Hand1, MSX1 and ectoderm marker Msl1 were all highly expressed in the EB samples while their expression was either undetectable or at low level in the ES samples.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	G3PDH was a positive control showing similar amount of RNA samples were used for analysis.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	We have also examined the cross-reactivities of these antibodies to mouse ES cells using mouse D3 ES cell line and mouse fetal endodermal tissue.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Cross-reactivity to mouse of goat anti-Oct3/4, goat anti-PDX-1, goat anti-SOX17 and mouse anti-SOX2 was detected.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Minimal cross-reactivity to mouse, measured by 10% intensity to human by higher than control cells, was observed in mouse anti-CD9 and mouse anti-E-cadherin antibodies.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Goat anti-Nanog and mouse anti-PODXL antibodies appear to be human-specific as well (data not shown).
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	The subtypes of monoclonal antibodies were also identified in the best clones.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	These results are summarized in Table 2.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Summary of antibodies detection in ES and EB samples. *
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	NT, Not tested; ND, Not determined.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	The expression patterns detected using antibodies developed in our facility are consistent with data reported using reverse transcriptase-polymerase chain reaction or cDNA microarrays.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Moreover several of the monoclonal antibodies have differing heavy chain subunits allowing double labeling using subtype specific markers to be performed.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	In summary, we have developed a useful collection of antibodies that would be useful for identification of stem cell characteristics and assessment of differentiation.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Several additional antibodies to the molecules that have been identified as potential cell lineage markers are currently under development using the same approach.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Brachyury (aa.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	1–202), DPPA5 (a.a.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	1–116), GATA1 (a.a.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	1–413), GATA6 (aa.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	1–449), Nanog (aa.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	153–305), Oct3/4 (aa.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	1–265), PDX-1 (aa.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	1–283), SOX2 (aa.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	135–317) and SOX17 (aa.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	177–414) were expressed in E. Coli and extracellular domains of CD9, E-Cadherin, PODXL were expressed in mouse NSO cells.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	All proteins were purified and sequenced before they were used as antigens for immunizations and as substrate for antibody screening and subcloning.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	All monoclonal antibodies were derived from fusions of mouse myeloma with B cells obtained from BALB/c mice which had been immunized with purified antigen.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	The IgG fraction of the culture supernatant was purified by Protein G affinity chromatography (Sigma).
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Each panel of antibodies was screened and selected for their abilities to detect purified recombinant antigen in direct ELISA and Western blot.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	All polyclonal antibodies were derived from sera of goats which had been immunized and boost it with purified antigen.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Antibody was purified from the sera by an antigen-affinity chromatography.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Human Caco-2, MG-63, MCF-7, NTERA-2 and mouse D3 cells were purchased from American Type Culture Collection (ATCC).
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Cells were cultured according to the ATCC instructions.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Information regarding human ES cell line HSF-6 (NIH code UC06) can be obtained at the website .
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Undifferentiated human ES cells were cultured according to the protocol provided by the University of California, San Francisco in human ES culture medium [DMEM supplemented with 20% KnockOut Serum Replacement (Invitrogen) and 5 ng/mL of bFGF (R&D Systems)].
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	To induce formation of embryoid bodies (EBs), ES colonies were harvested, separated from the MEF feeder cells by gravity, gently resuspended in ES culture medium and transferred to non-adherent suspension culture dishes (Corning).
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Unless otherwise noted, EBs derived from human ES cell aggregates were cultured for 8 days in ES culture medium deprived of bFGF and used for analysis by immunohistochemistry as described.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Cells are solubilized in hot 2× SDS gel sample buffer (20 mM dithiothreitol, 6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol, 10 mM NaF and bromophenyl blue) at 2 × 10per mL. The extracts are heated in a boiling water bath for 5 minutes and sonicated with a probe sonicator with 3–4 bursts of 5–10 seconds each.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Samples are diluted with 1× SDS sample buffer to the desired loading of 1–5 × 10per lane.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Lysates were resolved by SDS-PAGE, transferred to Immobilon-P membrane, and immunoblotted with 0.5 μg/mL primary Abs as described in R&D Systems Website .
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Antibodies were used with the appropriate secondary reagents at a concentration of 5 to 10 μg/ml.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Cells or sections of EBs were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min, then blocked and permeabilized with 0.1% Triton X-100, 1% BSA, 10% normal donkey serum in PBS at room temperature for 45 min.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	After blocking, cells were incubated with diluted primary antibody overnight at 4°C followed by coupled anti-mouse or anti-goat IgG (Molecular Probes) at room temperature in the dark for an hour.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Between each step cells were washed with PBS with 0.1% BSA.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Total RNA was extracted from EBs using Trizol LS (Invitrogen).
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	cDNA was synthesized by using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's recommendations.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	The PCR primers are available upon request.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Antibodies were prepared at the concentration of 0.1 mg/mL. 10 μL of the stock solution was added to 1 – 2.5 × 10cells in a total reaction volume not exceeding 200 μL. The sample was then incubated for 20 min at 2–8 °C.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Following incubation, excess antibody was removed by washing cells twice with FACS buffer (2% FCS and 0.1% sodium azide in Hank's buffer).
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	After wash, cells were resuspend in 200 μL of FACS buffer and the binding of unlabeled monoclonal antibodies was visualized by adding 10 μL of a 25 μg/mL stock solution of a secondary developing reagent such as goat anti-mouse IgG conjugated to a fluorochrome for 20 min at 2–8°C.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Following incubation, cells were washed once with FACS buffer, once with PBS.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	After wash, cells were resuspend in 400 μL of PBS and analyzed on a FACScant flow cytometer (Becton-Dickinson, Mountain View, CA).
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Five thousand events were collected and analyzed using CELL Quest software.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Dr. Cai contributed significantly in validating antibodies in human ES cells and human EBs.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Ms. Olson performed initial screening of antibodies in various cell lines.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Dr. Rao initiated the project, supervised Dr. Cai, and participated in all discussions for this report.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Ms. Stanley and Ms. Taylor performed Western blot analysis.
PMC1315352	Development of antibodies to human embryonic stem cell antigens.	Dr. Ni coordinated collaborative work between two labs, monitored the generation of the antibodies, and directed the project at R&D Systems.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Many novel studies and therapies are possible with the use of human embryonic stem cells (hES cells) and their differentiated cell progeny.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	The hES cell derived CD34 hematopoietic stem cells can be potentially used for many gene therapy applications.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages as a first step towards using these cells in viral infection studies and in developing novel stem cell based gene therapy strategies for AIDS.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Undifferentiated normal and lentiviral vector transduced hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34 hematopoietic progenitor cells.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	The differentiated CD34 cells isolated from cystic bodies were further cultured in cytokine media to derive macrophages.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Phenotypic and functional analyses were carried out to compare these with that of fetal liver CD34 cell derived macrophages.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	As assessed by FACS analysis, the hES-CD34 cell derived macrophages displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR suggesting a normal phenotype.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Tests evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation showed that these macrophages are also functionally normal.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	When infected with HIV-1, the differentiated macrophages supported productive viral infection.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Lentiviral vector transduced hES cells expressing the transgene GFP were evaluated similarly like above.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	The transgenic hES cells also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Phenotypically normal and functionally competent macrophages could be derived from hES-CD34 cells.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Since these cells are susceptible to HIV-1 infection, they provide a uniform source of macrophages for viral infection studies.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Based on these results, it is also now feasible to transduce hES-CD34 cells with anti-HIV genes such as inhibitory siRNAs and test their antiviral efficacy in down stream differentiated cells such as macrophages which are among the primary cells that need to be protected against HIV-1 infection.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Thus, the potential utility of hES derived CD34 hematopoietic cells for HIV-1 gene therapy can be evaluated.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Human embryonic stem cells (hES cells) show great promise for many novel cellular therapies due to their pluripotent nature .
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	These cells have the capacity to give rise to mature cells and tissues that arise from all three germ layers during embryonic development [2-4].
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Several pluripotent hES cell lines have so far been derived from the inner cell mass of human blastocysts and can be cultured indefinitely in an undifferentiated state [5-7].
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Thus, these cells provide a renewable source of pluripotent stem cells from which many types of differentiated cells could be produced for experimental and therapeutic purposes.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Cell differentiation protocols currently exist for the derivation of neurons, cardiomyocytes, endothelial cells, hematopoietic progenitor cells, keratinocytes, osteoblasts, and hepatocytes to name a few .
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	In addition to providing for potential cellular replacement therapies, opportunities exist in programming hES cells to correct a genetic defect and/or to express a therapeutic transgene of interest.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Using such approaches, many possibilities exist for treating a number of genetic and immune system disorders .
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Many novel applications can be foreseen for hES cells in infectious disease research.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	AIDS is a potential disease that can benefit from exploiting hES cells for cell replacement therapy as they have the capacity to differentiate into various hematopoietic cells.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	HIV continues to be a major global public health problem with infections increasing at an alarming rate .
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Given the present lack of effective vaccines and the ineffectiveness of drug based therapies for a complete cure, new and innovative approaches are essential.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Gene therapy through intracellular immunization offers a promising alternative approach and possible supplement to current HAART therapy [12-14].
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	HIV mainly targets cells of the hematopoietic system, namely, T cells, macrophages, and dendritic cells .
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	As infection progresses, the immune system is rendered defenseless against other invading pathogens and succumbs to opportunistic infections.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	There is a great deal of progress in the area of stem cell gene therapy for AIDS .
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	A primary goal of many ongoing studies is to introduce an effective anti-HIV gene into hematopoietic stem cells [16-18].
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	As these cells possess the ability to self renew, they have the potential to continually produce HIV resistant T cells and macrophages in the body thus providing long term immune reconstitution.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	These approaches use CD34 hematopoietic stem cells for anti-HIV gene transduction via integrating viral vectors such as lentiviral vectors [16-18].
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Lentiviral vectors have several advantages over conventional retroviral vectors since higher transduction efficiencies can be obtained and there is less gene silencing.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	The CD34 cells currently used for many therapies are primarily obtained from bone marrow or mobilized peripheral blood .
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Thus, CD34 progenitor cells are an essential ingredient for HIV gene therapy.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	In view of the need for CD34 cells for HIV gene therapy as well as for other hematopoietic disorders, if one can produce these cells in unlimited quantities from a renewable source, it will overcome the limitations of securing large numbers of CD34 cells for therapeutic purposes.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	In this regard, progress has been made in deriving CD34 cells from hES cells (hES-CD34).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Different methods currently exist to derive CD34 cells from hES cells with varying efficiencies [20-27].
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Recent reports have indicated the capacity of hES cell derived CD34 cells to give rise to lymphoid and myeloid lineages thus paving the way for utilization of these cells for hematopoietic cell therapy [20,27-29].
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	For the effective utilization of hES-CD34 cells for HIV gene therapy, a number of parameters need to be examined.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	First, one has to demonstrate that hES-CD34 cells can give rise to macrophages and helper T cells which are the main cells that need to be protected against HIV infection.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

E) The differentiation status of EB is detected by RT-PCR using different germ layer cell markers.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Selected endoderm markers AFP, FoxA2; mesoderm markers Hand1, MSX1 and ectoderm marker Msl1 were all highly expressed in the EB samples while their expression was either undetectable or at low level in the ES samples.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

G3PDH was a positive control showing similar amount of RNA samples were used for analysis.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

We have also examined the cross-reactivities of these antibodies to mouse ES cells using mouse D3 ES cell line and mouse fetal endodermal tissue.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Cross-reactivity to mouse of goat anti-Oct3/4, goat anti-PDX-1, goat anti-SOX17 and mouse anti-SOX2 was detected.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Minimal cross-reactivity to mouse, measured by 10% intensity to human by higher than control cells, was observed in mouse anti-CD9 and mouse anti-E-cadherin antibodies.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Goat anti-Nanog and mouse anti-PODXL antibodies appear to be human-specific as well (data not shown).

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

The subtypes of monoclonal antibodies were also identified in the best clones.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

These results are summarized in Table 2.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Summary of antibodies detection in ES and EB samples. *

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

NT, Not tested; ND, Not determined.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

The expression patterns detected using antibodies developed in our facility are consistent with data reported using reverse transcriptase-polymerase chain reaction or cDNA microarrays.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Moreover several of the monoclonal antibodies have differing heavy chain subunits allowing double labeling using subtype specific markers to be performed.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

In summary, we have developed a useful collection of antibodies that would be useful for identification of stem cell characteristics and assessment of differentiation.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Several additional antibodies to the molecules that have been identified as potential cell lineage markers are currently under development using the same approach.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Brachyury (aa.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

1–202), DPPA5 (a.a.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

1–116), GATA1 (a.a.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

1–413), GATA6 (aa.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

1–449), Nanog (aa.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

153–305), Oct3/4 (aa.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

1–265), PDX-1 (aa.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

1–283), SOX2 (aa.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

135–317) and SOX17 (aa.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

177–414) were expressed in E. Coli and extracellular domains of CD9, E-Cadherin, PODXL were expressed in mouse NSO cells.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

All proteins were purified and sequenced before they were used as antigens for immunizations and as substrate for antibody screening and subcloning.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

All monoclonal antibodies were derived from fusions of mouse myeloma with B cells obtained from BALB/c mice which had been immunized with purified antigen.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

The IgG fraction of the culture supernatant was purified by Protein G affinity chromatography (Sigma).

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Each panel of antibodies was screened and selected for their abilities to detect purified recombinant antigen in direct ELISA and Western blot.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

All polyclonal antibodies were derived from sera of goats which had been immunized and boost it with purified antigen.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Antibody was purified from the sera by an antigen-affinity chromatography.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Human Caco-2, MG-63, MCF-7, NTERA-2 and mouse D3 cells were purchased from American Type Culture Collection (ATCC).

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Cells were cultured according to the ATCC instructions.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Information regarding human ES cell line HSF-6 (NIH code UC06) can be obtained at the website .

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Undifferentiated human ES cells were cultured according to the protocol provided by the University of California, San Francisco in human ES culture medium [DMEM supplemented with 20% KnockOut Serum Replacement (Invitrogen) and 5 ng/mL of bFGF (R&D Systems)].

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

To induce formation of embryoid bodies (EBs), ES colonies were harvested, separated from the MEF feeder cells by gravity, gently resuspended in ES culture medium and transferred to non-adherent suspension culture dishes (Corning).

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Unless otherwise noted, EBs derived from human ES cell aggregates were cultured for 8 days in ES culture medium deprived of bFGF and used for analysis by immunohistochemistry as described.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Cells are solubilized in hot 2× SDS gel sample buffer (20 mM dithiothreitol, 6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol, 10 mM NaF and bromophenyl blue) at 2 × 10per mL. The extracts are heated in a boiling water bath for 5 minutes and sonicated with a probe sonicator with 3–4 bursts of 5–10 seconds each.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Samples are diluted with 1× SDS sample buffer to the desired loading of 1–5 × 10per lane.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Lysates were resolved by SDS-PAGE, transferred to Immobilon-P membrane, and immunoblotted with 0.5 μg/mL primary Abs as described in R&D Systems Website .

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Antibodies were used with the appropriate secondary reagents at a concentration of 5 to 10 μg/ml.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Cells or sections of EBs were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min, then blocked and permeabilized with 0.1% Triton X-100, 1% BSA, 10% normal donkey serum in PBS at room temperature for 45 min.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

After blocking, cells were incubated with diluted primary antibody overnight at 4°C followed by coupled anti-mouse or anti-goat IgG (Molecular Probes) at room temperature in the dark for an hour.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Between each step cells were washed with PBS with 0.1% BSA.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Total RNA was extracted from EBs using Trizol LS (Invitrogen).

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

cDNA was synthesized by using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's recommendations.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

The PCR primers are available upon request.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Antibodies were prepared at the concentration of 0.1 mg/mL. 10 μL of the stock solution was added to 1 – 2.5 × 10cells in a total reaction volume not exceeding 200 μL. The sample was then incubated for 20 min at 2–8 °C.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Following incubation, excess antibody was removed by washing cells twice with FACS buffer (2% FCS and 0.1% sodium azide in Hank's buffer).

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

After wash, cells were resuspend in 200 μL of FACS buffer and the binding of unlabeled monoclonal antibodies was visualized by adding 10 μL of a 25 μg/mL stock solution of a secondary developing reagent such as goat anti-mouse IgG conjugated to a fluorochrome for 20 min at 2–8°C.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Following incubation, cells were washed once with FACS buffer, once with PBS.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

After wash, cells were resuspend in 400 μL of PBS and analyzed on a FACScant flow cytometer (Becton-Dickinson, Mountain View, CA).

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Five thousand events were collected and analyzed using CELL Quest software.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Dr. Cai contributed significantly in validating antibodies in human ES cells and human EBs.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Ms. Olson performed initial screening of antibodies in various cell lines.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Dr. Rao initiated the project, supervised Dr. Cai, and participated in all discussions for this report.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Ms. Stanley and Ms. Taylor performed Western blot analysis.

PMC1315352

Development of antibodies to human embryonic stem cell antigens.

Dr. Ni coordinated collaborative work between two labs, monitored the generation of the antibodies, and directed the project at R&D Systems.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Many novel studies and therapies are possible with the use of human embryonic stem cells (hES cells) and their differentiated cell progeny.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

The hES cell derived CD34 hematopoietic stem cells can be potentially used for many gene therapy applications.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Here we evaluated the capacity of hES cell derived CD34 cells to give rise to normal macrophages as a first step towards using these cells in viral infection studies and in developing novel stem cell based gene therapy strategies for AIDS.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Undifferentiated normal and lentiviral vector transduced hES cells were cultured on S17 mouse bone marrow stromal cell layers to derive CD34 hematopoietic progenitor cells.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

The differentiated CD34 cells isolated from cystic bodies were further cultured in cytokine media to derive macrophages.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Phenotypic and functional analyses were carried out to compare these with that of fetal liver CD34 cell derived macrophages.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

As assessed by FACS analysis, the hES-CD34 cell derived macrophages displayed characteristic cell surface markers CD14, CD4, CCR5, CXCR4, and HLA-DR suggesting a normal phenotype.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Tests evaluating phagocytosis, upregulation of the costimulatory molecule B7.1, and cytokine secretion in response to LPS stimulation showed that these macrophages are also functionally normal.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

When infected with HIV-1, the differentiated macrophages supported productive viral infection.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Lentiviral vector transduced hES cells expressing the transgene GFP were evaluated similarly like above.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

The transgenic hES cells also gave rise to macrophages with normal phenotypic and functional characteristics indicating no vector mediated adverse effects during differentiation.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Phenotypically normal and functionally competent macrophages could be derived from hES-CD34 cells.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Since these cells are susceptible to HIV-1 infection, they provide a uniform source of macrophages for viral infection studies.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Based on these results, it is also now feasible to transduce hES-CD34 cells with anti-HIV genes such as inhibitory siRNAs and test their antiviral efficacy in down stream differentiated cells such as macrophages which are among the primary cells that need to be protected against HIV-1 infection.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Thus, the potential utility of hES derived CD34 hematopoietic cells for HIV-1 gene therapy can be evaluated.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Human embryonic stem cells (hES cells) show great promise for many novel cellular therapies due to their pluripotent nature .

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

These cells have the capacity to give rise to mature cells and tissues that arise from all three germ layers during embryonic development [2-4].

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Several pluripotent hES cell lines have so far been derived from the inner cell mass of human blastocysts and can be cultured indefinitely in an undifferentiated state [5-7].

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Thus, these cells provide a renewable source of pluripotent stem cells from which many types of differentiated cells could be produced for experimental and therapeutic purposes.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Cell differentiation protocols currently exist for the derivation of neurons, cardiomyocytes, endothelial cells, hematopoietic progenitor cells, keratinocytes, osteoblasts, and hepatocytes to name a few .

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

In addition to providing for potential cellular replacement therapies, opportunities exist in programming hES cells to correct a genetic defect and/or to express a therapeutic transgene of interest.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Using such approaches, many possibilities exist for treating a number of genetic and immune system disorders .

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Many novel applications can be foreseen for hES cells in infectious disease research.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

AIDS is a potential disease that can benefit from exploiting hES cells for cell replacement therapy as they have the capacity to differentiate into various hematopoietic cells.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

HIV continues to be a major global public health problem with infections increasing at an alarming rate .

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Given the present lack of effective vaccines and the ineffectiveness of drug based therapies for a complete cure, new and innovative approaches are essential.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Gene therapy through intracellular immunization offers a promising alternative approach and possible supplement to current HAART therapy [12-14].

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

HIV mainly targets cells of the hematopoietic system, namely, T cells, macrophages, and dendritic cells .

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

As infection progresses, the immune system is rendered defenseless against other invading pathogens and succumbs to opportunistic infections.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

There is a great deal of progress in the area of stem cell gene therapy for AIDS .

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

A primary goal of many ongoing studies is to introduce an effective anti-HIV gene into hematopoietic stem cells [16-18].

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

As these cells possess the ability to self renew, they have the potential to continually produce HIV resistant T cells and macrophages in the body thus providing long term immune reconstitution.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

These approaches use CD34 hematopoietic stem cells for anti-HIV gene transduction via integrating viral vectors such as lentiviral vectors [16-18].

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Lentiviral vectors have several advantages over conventional retroviral vectors since higher transduction efficiencies can be obtained and there is less gene silencing.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

The CD34 cells currently used for many therapies are primarily obtained from bone marrow or mobilized peripheral blood .

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Thus, CD34 progenitor cells are an essential ingredient for HIV gene therapy.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

In view of the need for CD34 cells for HIV gene therapy as well as for other hematopoietic disorders, if one can produce these cells in unlimited quantities from a renewable source, it will overcome the limitations of securing large numbers of CD34 cells for therapeutic purposes.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

In this regard, progress has been made in deriving CD34 cells from hES cells (hES-CD34).

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Different methods currently exist to derive CD34 cells from hES cells with varying efficiencies [20-27].

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

Recent reports have indicated the capacity of hES cell derived CD34 cells to give rise to lymphoid and myeloid lineages thus paving the way for utilization of these cells for hematopoietic cell therapy [20,27-29].

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

For the effective utilization of hES-CD34 cells for HIV gene therapy, a number of parameters need to be examined.

PMC1462997

Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.

First, one has to demonstrate that hES-CD34 cells can give rise to macrophages and helper T cells which are the main cells that need to be protected against HIV infection.