Mardiyyah/TAPT_data_V2_split · Datasets at Hugging Face

PMCID stringclasses 24 values	Title stringclasses 24 values	Sentences stringlengths 2 40.7k
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Recent evidence has shown that hES-CD34 cells can give rise to myelomonocytic cells .
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	However, thorough phenotypic or functional characterization of these cells is lacking.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	It is also not clear if these cells are susceptible to HIV infection.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Similarly, although the hES-CD34 cells were shown to have lymphoid progenitor capacity, only B cell and natural killer (NK) cell differentiation has been examined so far .
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	The capacity to generate T cells remains to be evaluated.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	With this background, as a first step, our primary goal in these studies is to examine the capacity of hES-CD34 cells to give rise to phenotypically and functionally normal macrophages and whether such cells are susceptible to productive HIV infection.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Since lentiviral vectors have been shown to successfully transduce hES cells [30-33], we further investigated the ability of transduced hES cells to differentiate into transgenic macrophages that can support HIV-1 infection.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Demonstration of HIV-1 productive infection in these cells will permit future efficacy evaluations of anti-HIV genes in this system.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Here we show that normal and lentiviral vector transduced hES-CD34 cells can give rise to phenotypically and functionally normal macrophages that support HIV infection thus paving the way for many novel approaches to evaluate their potential for HIV gene therapy.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Undifferentiated hES cell colonies grown in media supplemented with 4 ng/ml bFGF displayed normal morphology of pluripotent human embryonic stem cells with tight and discreet borders on the MEF feeder layers (Fig 1A).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Similarly, lentiviral vector transduced hES cell colonies, also displayed normal morphology and growth characteristics (Fig 1A).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	As expected, the vector transduced colonies displayed green fluorescence due to the presence of the GFP reporter gene.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	When cultured on irradiated S17 mouse bone marrow stromal cells, both nontransduced and transduced hES cells developed into embryonic cystic bodies (Fig 1A).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	FACS analysis of single cell suspensions of the cystic bodies showed levels of CD34 cells which ranged from 7–15%.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Figure 1B displays a representative FACS profile of hES-CD34 cells.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Purified CD34 cells were later cultured in semi-solid methylcellulose medium to derive myeloid colonies.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Both nontransduced (denoted as ES in figures) and vector transduced (denoted as GFP ES in figures) hES cell derived CD34 cells gave rise to normal myelomonocytic colonies similar to human fetal liver derived CD34 cells (denoted as CD34 in figures) (Fig 1A).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	When pooled colonies were cultured further in liquid cytokine media for 12–15 days for differentiation, the cells developed into morphologically distinct macrophages (Fig 1A).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	When compared, the morphology of macrophages derived from all stem cell progenitor populations appeared similar.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	These results were found to be consistent in replicative experiments.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	The transgene GFP expression was also maintained during the differentiation of hES cells into mature macrophages.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	GFP expression in cystic body derived CD34 cells was around 80% (data not shown) with similar levels seen in differentiated macrophages (Fig 2).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Derivation of macrophages from lentiviral vector transduced and normal hES cells.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	A) Transduced and non-transduced H1 hES cells were cultured on mouse S17 bone marrow stromal cell layers to derive cystic bodies.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Cystic body derived CD34 cells were purified by positive selection with antibody conjugated magnetic beads and placed in methocult media to obtain myelomonocytic colonies.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Pooled colonies were cultured in liquid cytokine media supplemented with GM-CSF and M-CSF to promote macrophage growth.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	For comparison, fetal liver derived CD34 cells were cultured similarly to derive macrophages.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Representative ES cell colonies, cystic bodies, methocult colonies, and derivative macrophages are shown with GFP expressing cells fluorescing green under UV illumination.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	B) Representative FACS profile of hES cell derived CD34 cells stained with PE conjugated antibodies.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Percent positive CD34 cells are shown with isotype control shown in the left panel.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Phenotypic FACS analysis of hES cell derived macrophages.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	A) Macrophages derived from transduced and nontransduced hES CD34 and fetal liver CD34 cells were stained with antibodies to CD14, HLA-DR, CD4, CCR5, and CXCR4 and the expression of these surface markers was analyzed by FACS.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	B) Isotype controls for PE and PE-CY5 antibodies.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Percent positive cells are displayed in the plots for each respective cell surface marker staining.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Dot plots are representative of triplicate experiments.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Macrophages play a critical role in immune system function and are also major target cells for many viral infections including HIV-1.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Distinct surface phenotypic markers exist on these cells and, thus far, there has been no thorough evaluation of hES cell derived macrophages.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Therefore we analyzed hES cell derived macrophages for the presence of characteristic cell surface markers and compared these to the phenotypic profile displayed on fetal CD34 cell derived macrophages.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	The surface markers analyzed were CD14, a monocyte/macrophage specific marker, HLA-DR (a class II MHC molecule found on antigen presenting cells), CD4, the major receptor for HIV-1 infection, and CCR5 and CXCR4, chemokine receptors which are critical coreceptors essential for HIV-1 entry.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	EGFP expression was also analyzed to determine the levels of transduction and any transgene silencing that may occur during differentiation.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Fetal liver (CD34), nontransduced (ES), and vector transduced (GFP ES) hES cell derived macrophages were all positive for the monocyte/macrophage marker CD14 (99.3%, 88.7%, and 99.2%, respectively) (Fig 2A).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	However, the mean fluorescent intensity (MFI) was found to be lower on hES cell derived macrophages.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Surface expression of HLA-DR was observed at similar levels between macrophages derived from fetal liver CD34 cells (99.6%), nontransduced hES cells (92.8%), and transduced hES cells (98.2%) (Fig 2A).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	CD4 levels were comparable for all stem cell derived macrophages (99.2%, 83.3%, and 88.7%, respectively) (Fig 2A).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	CCR5 and CXCR4 cell surface expression was also observed for fetal liver CD34 cell (99.6% and 99.3%), nontransduced hES cell (91.9% and 92.6%), and transduced hES cell (98.9% and 99.3%) derived macrophages (Fig 2A).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	As compared to fetal liver CD34 cell derived macrophages, hES cell derived macrophages displayed a higher level of expression of CXCR4.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Isotype controls for both PE and PECY5 stains are shown in Fig 2B.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	The above phenotypic data are representative of triplicate experiments.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	The antigen presenting cell surface specific marker HLA-DR (MHC II) on normal macrophages is critical for presenting antigen to CD4 T cells.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	A second co-stimulatory molecule, B7.1 is present at low basal levels on resting macrophages and is necessary to activate T cells.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Its expression is elevated upon activation with certain stimuli such as LPS.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Our results of LPS stimulation of respective macrophages have shown upregulation of B7.1 with values for fetal liver CD34 cell (CD34) (27.9% to 75.4%) nontransduced (ES) (17.8% to 49.4%) and transduced (GFP ES) (35.6% to 65.7%) hES cell derived macrophages (Fig 3A).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	These values represent a significant upregulation of B7.1 for all three macrophage populations.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Functional analysis of hES cell derived macrophages for B7.1 costimulatory molecule upregulation and phagocytosis of E. coli particles: A) Mature macrophages were stimulated with LPS to determine B7.1 upregulation.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Twenty-four hours post-stimulation, macrophages were labeled with a PE-CY5 conjugated anti-B7.1 antibody and analyzed by FACS.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	B7.1 upregulation data are representative of triplicate experiments.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Isotype control is shown in the left panel.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	B) To assess phagocytic function, E. coli Bioparticleswere added directly to the cultured macrophages.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Twenty four hours post-addition, cells were analyzed by FACS.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Percent positive cells are displayed in the plots for each experiment.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	These data are representative of triplicate experiments.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Another important function of macrophages is their ability to phagocytose foreign material and present antigenic peptides on their cell surface.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	To evaluate phagocytic function, fluorescently labeled E. coli Bioparticleswere added to macrophage cultures followed by FACS analysis.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Nontransduced (94.6%) as well as lentiviral vector transduced (98.7%) hES cell derived macrophages were found to be capable of phagocytosing the Bioparticlesin comparison to fetal liver CD34 cell derived macrophages (95.8%) (Fig 3B).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	These values are representative of triplicate experiments.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Magi-CXCR4 cells with no phagocytic capacity were used as non-phagocytic cell controls and similarly exposed to E. coli Bioparticles(Fig 3B).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	No uptake of the bacteria could be seen.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Thus, uptake of E. coli Bioparticlesby macrophages is indicative of active ingestion.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Macrophages, as effector cells, play a key role in the inflammatory response.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Activated macrophages secrete various cytokines, two of the major ones being IL-1 and TNF-α.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	To determine if hES cell derived macrophages have such a capacity, cells were stimulated with LPS.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	On days 1, 2, and 3 post-stimulation, culture supernatants were analyzed by ELISA to detect IL-1 and TNF-α.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	As seen in figure 4A, there were no significant differences in IL-1 secretion between the three sets of macrophages.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Similarly, nontransduced and transduced hES cell derived macrophages were also capable of TNF-α secretion upon LPS stimulation.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	However, levels of the respective cytokines detected were slightly lower than those from fetal liver CD34 cell derived macrophages (Fig 4B).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	The values of cytokine secretion levels represent triplicate experiments.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Cytokine IL-1 and TNFα secretion by stimulated hES cell derived macrophages: Macrophages derived from transduced and nontransduced hES and fetal liver CD34 cells were stimulated with 5 μg/ml LPS.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	On days 1, 2, and 3 post-stimulation, supernatants were collected and assayed by ELISA for (A) IL-1 and (B) TNFα.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Experiments were done in triplicate.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	The above data have shown that hES cell derived macrophages are very similar to normal human macrophages based on phenotypic and functional analysis.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	In addition to being important cells of the immune system, macrophages are among the major target cells for certain viral infections, particularly for HIV-1.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	We wanted to determine if hES cell derived macrophages were susceptible to HIV-1 infection compared to standard macrophages.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	In these studies, we only used an R5-tropic strain of HIV-1 since macrophages are natural targets for this virus.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Our results from challenge studies of these cells clearly indicated the capacity of hES cell derived macrophages in supporting a productive infection.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Levels of virus increased up to 15 days similar to non-hES derived macrophages showing that the initial viral input was amplified in productive viral infection.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	However, the levels of viral yield were found to be slightly lower for the ES cell derived macrophages.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	In the case of GFP-ES macrophages, there was a decline in viral titer.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	This could be due to possible lower numbers of cells present in the initial cultures.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	As a first step towards the use of hES cells for hematopoietic stem cell and HIV gene therapies, we have shown here that phenotypically and functionally normal 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.	Both non transduced and lentiviral vector transduced hES cells were found to be capable of generating CD34 cells that give rise to macrophages which could support productive HIV-1 infection.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Current sources of CD34 cells consist of human bone marrow, cytokine mobilized peripheral blood, fetal liver, and cord blood .
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	However, the number of cells that can be obtained for manipulations is not unlimited.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Therefore, deriving CD34 cells for therapeutic and investigative purposes from hES cells with unlimited growth potential has the advantage of a consistent and uniform source.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	The ability to obtain phenotypically normal and functionally competent macrophages from hES cells is important to evaluate their potential therapeutic utilities in the future.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Additionally, testing of transgenic hES cells derived via lentiviral vector gene transduction is also helpful to determine the stability of the transgene expression and their capacity for differentiation into end stage mature cells such as macrophages.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Based on these considerations, both non- transduced and lentiviral vector transduced hES cells were evaluated for their capacity to give rise to CD34 progenitor cells.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	In colony forming assays using semisolid methylcellulose medium, the morphology of myelomonocytic colonies derived from hES CD34 cells appeared similar to that of fetal liver CD34 cells.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	When subsequently cultured in cytokine media that promotes macrophage differentiation, morphologically normal macrophages were obtained with hES-CD34 cells similar to that of fetal liver CD34 cells.
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	At higher magnification, the macrophages displayed flat projecting cellular borders with fried egg appearance with distinct refractory lysosomal granules in the cytoplasm (data not shown).
PMC1462997	Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.	Lentiviral vector transduced hES cells also did not display any abnormal growth or differentiation characteristics as compared to nontransduced hES-CD34 cells indicating no adverse effects due to vector integration and expression.

PMC1462997

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

Recent evidence has shown that hES-CD34 cells can give rise to myelomonocytic cells .

PMC1462997

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

However, thorough phenotypic or functional characterization of these cells is lacking.

PMC1462997

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

It is also not clear if these cells are susceptible to HIV infection.

PMC1462997

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

Similarly, although the hES-CD34 cells were shown to have lymphoid progenitor capacity, only B cell and natural killer (NK) cell differentiation has been examined so far .

PMC1462997

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

The capacity to generate T cells remains to be evaluated.

PMC1462997

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

With this background, as a first step, our primary goal in these studies is to examine the capacity of hES-CD34 cells to give rise to phenotypically and functionally normal macrophages and whether such cells are susceptible to productive HIV infection.

PMC1462997

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

Since lentiviral vectors have been shown to successfully transduce hES cells [30-33], we further investigated the ability of transduced hES cells to differentiate into transgenic macrophages that can support HIV-1 infection.

PMC1462997

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

Demonstration of HIV-1 productive infection in these cells will permit future efficacy evaluations of anti-HIV genes in this system.

PMC1462997

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

Here we show that normal and lentiviral vector transduced hES-CD34 cells can give rise to phenotypically and functionally normal macrophages that support HIV infection thus paving the way for many novel approaches to evaluate their potential for HIV gene therapy.

PMC1462997

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

Undifferentiated hES cell colonies grown in media supplemented with 4 ng/ml bFGF displayed normal morphology of pluripotent human embryonic stem cells with tight and discreet borders on the MEF feeder layers (Fig 1A).

PMC1462997

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

Similarly, lentiviral vector transduced hES cell colonies, also displayed normal morphology and growth characteristics (Fig 1A).

PMC1462997

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

As expected, the vector transduced colonies displayed green fluorescence due to the presence of the GFP reporter gene.

PMC1462997

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

When cultured on irradiated S17 mouse bone marrow stromal cells, both nontransduced and transduced hES cells developed into embryonic cystic bodies (Fig 1A).

PMC1462997

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

FACS analysis of single cell suspensions of the cystic bodies showed levels of CD34 cells which ranged from 7–15%.

PMC1462997

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

Figure 1B displays a representative FACS profile of hES-CD34 cells.

PMC1462997

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

Purified CD34 cells were later cultured in semi-solid methylcellulose medium to derive myeloid colonies.

PMC1462997

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

Both nontransduced (denoted as ES in figures) and vector transduced (denoted as GFP ES in figures) hES cell derived CD34 cells gave rise to normal myelomonocytic colonies similar to human fetal liver derived CD34 cells (denoted as CD34 in figures) (Fig 1A).

PMC1462997

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

When pooled colonies were cultured further in liquid cytokine media for 12–15 days for differentiation, the cells developed into morphologically distinct macrophages (Fig 1A).

PMC1462997

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

When compared, the morphology of macrophages derived from all stem cell progenitor populations appeared similar.

PMC1462997

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

These results were found to be consistent in replicative experiments.

PMC1462997

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

The transgene GFP expression was also maintained during the differentiation of hES cells into mature macrophages.

PMC1462997

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

GFP expression in cystic body derived CD34 cells was around 80% (data not shown) with similar levels seen in differentiated macrophages (Fig 2).

PMC1462997

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

Derivation of macrophages from lentiviral vector transduced and normal hES cells.

PMC1462997

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

A) Transduced and non-transduced H1 hES cells were cultured on mouse S17 bone marrow stromal cell layers to derive cystic bodies.

PMC1462997

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

Cystic body derived CD34 cells were purified by positive selection with antibody conjugated magnetic beads and placed in methocult media to obtain myelomonocytic colonies.

PMC1462997

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

Pooled colonies were cultured in liquid cytokine media supplemented with GM-CSF and M-CSF to promote macrophage growth.

PMC1462997

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

For comparison, fetal liver derived CD34 cells were cultured similarly to derive macrophages.

PMC1462997

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

Representative ES cell colonies, cystic bodies, methocult colonies, and derivative macrophages are shown with GFP expressing cells fluorescing green under UV illumination.

PMC1462997

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

B) Representative FACS profile of hES cell derived CD34 cells stained with PE conjugated antibodies.

PMC1462997

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

Percent positive CD34 cells are shown with isotype control shown in the left panel.

PMC1462997

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

Phenotypic FACS analysis of hES cell derived macrophages.

PMC1462997

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

A) Macrophages derived from transduced and nontransduced hES CD34 and fetal liver CD34 cells were stained with antibodies to CD14, HLA-DR, CD4, CCR5, and CXCR4 and the expression of these surface markers was analyzed by FACS.

PMC1462997

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

B) Isotype controls for PE and PE-CY5 antibodies.

PMC1462997

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

Percent positive cells are displayed in the plots for each respective cell surface marker staining.

PMC1462997

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

Dot plots are representative of triplicate experiments.

PMC1462997

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

Macrophages play a critical role in immune system function and are also major target cells for many viral infections including HIV-1.

PMC1462997

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

Distinct surface phenotypic markers exist on these cells and, thus far, there has been no thorough evaluation of hES cell derived macrophages.

PMC1462997

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

Therefore we analyzed hES cell derived macrophages for the presence of characteristic cell surface markers and compared these to the phenotypic profile displayed on fetal CD34 cell derived macrophages.

PMC1462997

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

The surface markers analyzed were CD14, a monocyte/macrophage specific marker, HLA-DR (a class II MHC molecule found on antigen presenting cells), CD4, the major receptor for HIV-1 infection, and CCR5 and CXCR4, chemokine receptors which are critical coreceptors essential for HIV-1 entry.

PMC1462997

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

EGFP expression was also analyzed to determine the levels of transduction and any transgene silencing that may occur during differentiation.

PMC1462997

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

Fetal liver (CD34), nontransduced (ES), and vector transduced (GFP ES) hES cell derived macrophages were all positive for the monocyte/macrophage marker CD14 (99.3%, 88.7%, and 99.2%, respectively) (Fig 2A).

PMC1462997

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

However, the mean fluorescent intensity (MFI) was found to be lower on hES cell derived macrophages.

PMC1462997

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

Surface expression of HLA-DR was observed at similar levels between macrophages derived from fetal liver CD34 cells (99.6%), nontransduced hES cells (92.8%), and transduced hES cells (98.2%) (Fig 2A).

PMC1462997

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

CD4 levels were comparable for all stem cell derived macrophages (99.2%, 83.3%, and 88.7%, respectively) (Fig 2A).

PMC1462997

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

CCR5 and CXCR4 cell surface expression was also observed for fetal liver CD34 cell (99.6% and 99.3%), nontransduced hES cell (91.9% and 92.6%), and transduced hES cell (98.9% and 99.3%) derived macrophages (Fig 2A).

PMC1462997

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

As compared to fetal liver CD34 cell derived macrophages, hES cell derived macrophages displayed a higher level of expression of CXCR4.

PMC1462997

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

Isotype controls for both PE and PECY5 stains are shown in Fig 2B.

PMC1462997

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

The above phenotypic data are representative of triplicate experiments.

PMC1462997

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

The antigen presenting cell surface specific marker HLA-DR (MHC II) on normal macrophages is critical for presenting antigen to CD4 T cells.

PMC1462997

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

A second co-stimulatory molecule, B7.1 is present at low basal levels on resting macrophages and is necessary to activate T cells.

PMC1462997

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

Its expression is elevated upon activation with certain stimuli such as LPS.

PMC1462997

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

Our results of LPS stimulation of respective macrophages have shown upregulation of B7.1 with values for fetal liver CD34 cell (CD34) (27.9% to 75.4%) nontransduced (ES) (17.8% to 49.4%) and transduced (GFP ES) (35.6% to 65.7%) hES cell derived macrophages (Fig 3A).

PMC1462997

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

These values represent a significant upregulation of B7.1 for all three macrophage populations.

PMC1462997

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

Functional analysis of hES cell derived macrophages for B7.1 costimulatory molecule upregulation and phagocytosis of E. coli particles: A) Mature macrophages were stimulated with LPS to determine B7.1 upregulation.

PMC1462997

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

Twenty-four hours post-stimulation, macrophages were labeled with a PE-CY5 conjugated anti-B7.1 antibody and analyzed by FACS.

PMC1462997

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

B7.1 upregulation data are representative of triplicate experiments.

PMC1462997

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

Isotype control is shown in the left panel.

PMC1462997

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

B) To assess phagocytic function, E. coli Bioparticleswere added directly to the cultured macrophages.

PMC1462997

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

Twenty four hours post-addition, cells were analyzed by FACS.

PMC1462997

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

Percent positive cells are displayed in the plots for each experiment.

PMC1462997

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

These data are representative of triplicate experiments.

PMC1462997

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

Another important function of macrophages is their ability to phagocytose foreign material and present antigenic peptides on their cell surface.

PMC1462997

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

To evaluate phagocytic function, fluorescently labeled E. coli Bioparticleswere added to macrophage cultures followed by FACS analysis.

PMC1462997

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

Nontransduced (94.6%) as well as lentiviral vector transduced (98.7%) hES cell derived macrophages were found to be capable of phagocytosing the Bioparticlesin comparison to fetal liver CD34 cell derived macrophages (95.8%) (Fig 3B).

PMC1462997

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

These values are representative of triplicate experiments.

PMC1462997

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

Magi-CXCR4 cells with no phagocytic capacity were used as non-phagocytic cell controls and similarly exposed to E. coli Bioparticles(Fig 3B).

PMC1462997

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

No uptake of the bacteria could be seen.

PMC1462997

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

Thus, uptake of E. coli Bioparticlesby macrophages is indicative of active ingestion.

PMC1462997

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

Macrophages, as effector cells, play a key role in the inflammatory response.

PMC1462997

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

Activated macrophages secrete various cytokines, two of the major ones being IL-1 and TNF-α.

PMC1462997

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

To determine if hES cell derived macrophages have such a capacity, cells were stimulated with LPS.

PMC1462997

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

On days 1, 2, and 3 post-stimulation, culture supernatants were analyzed by ELISA to detect IL-1 and TNF-α.

PMC1462997

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

As seen in figure 4A, there were no significant differences in IL-1 secretion between the three sets of macrophages.

PMC1462997

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

Similarly, nontransduced and transduced hES cell derived macrophages were also capable of TNF-α secretion upon LPS stimulation.

PMC1462997

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

However, levels of the respective cytokines detected were slightly lower than those from fetal liver CD34 cell derived macrophages (Fig 4B).

PMC1462997

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

The values of cytokine secretion levels represent triplicate experiments.

PMC1462997

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

Cytokine IL-1 and TNFα secretion by stimulated hES cell derived macrophages: Macrophages derived from transduced and nontransduced hES and fetal liver CD34 cells were stimulated with 5 μg/ml LPS.

PMC1462997

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

On days 1, 2, and 3 post-stimulation, supernatants were collected and assayed by ELISA for (A) IL-1 and (B) TNFα.

PMC1462997

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

Experiments were done in triplicate.

PMC1462997

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

The above data have shown that hES cell derived macrophages are very similar to normal human macrophages based on phenotypic and functional analysis.

PMC1462997

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

In addition to being important cells of the immune system, macrophages are among the major target cells for certain viral infections, particularly for HIV-1.

PMC1462997

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

We wanted to determine if hES cell derived macrophages were susceptible to HIV-1 infection compared to standard macrophages.

PMC1462997

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

In these studies, we only used an R5-tropic strain of HIV-1 since macrophages are natural targets for this virus.

PMC1462997

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

Our results from challenge studies of these cells clearly indicated the capacity of hES cell derived macrophages in supporting a productive infection.

PMC1462997

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

Levels of virus increased up to 15 days similar to non-hES derived macrophages showing that the initial viral input was amplified in productive viral infection.

PMC1462997

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

However, the levels of viral yield were found to be slightly lower for the ES cell derived macrophages.

PMC1462997

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

In the case of GFP-ES macrophages, there was a decline in viral titer.

PMC1462997

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

This could be due to possible lower numbers of cells present in the initial cultures.

PMC1462997

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

As a first step towards the use of hES cells for hematopoietic stem cell and HIV gene therapies, we have shown here that phenotypically and functionally normal 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.

Both non transduced and lentiviral vector transduced hES cells were found to be capable of generating CD34 cells that give rise to macrophages which could support productive HIV-1 infection.

PMC1462997

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

Current sources of CD34 cells consist of human bone marrow, cytokine mobilized peripheral blood, fetal liver, and cord blood .

PMC1462997

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

However, the number of cells that can be obtained for manipulations is not unlimited.

PMC1462997

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

Therefore, deriving CD34 cells for therapeutic and investigative purposes from hES cells with unlimited growth potential has the advantage of a consistent and uniform source.

PMC1462997

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

The ability to obtain phenotypically normal and functionally competent macrophages from hES cells is important to evaluate their potential therapeutic utilities in the future.

PMC1462997

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

Additionally, testing of transgenic hES cells derived via lentiviral vector gene transduction is also helpful to determine the stability of the transgene expression and their capacity for differentiation into end stage mature cells such as macrophages.

PMC1462997

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

Based on these considerations, both non- transduced and lentiviral vector transduced hES cells were evaluated for their capacity to give rise to CD34 progenitor cells.

PMC1462997

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

In colony forming assays using semisolid methylcellulose medium, the morphology of myelomonocytic colonies derived from hES CD34 cells appeared similar to that of fetal liver CD34 cells.

PMC1462997

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

When subsequently cultured in cytokine media that promotes macrophage differentiation, morphologically normal macrophages were obtained with hES-CD34 cells similar to that of fetal liver CD34 cells.

PMC1462997

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

At higher magnification, the macrophages displayed flat projecting cellular borders with fried egg appearance with distinct refractory lysosomal granules in the cytoplasm (data not shown).

PMC1462997

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

Lentiviral vector transduced hES cells also did not display any abnormal growth or differentiation characteristics as compared to nontransduced hES-CD34 cells indicating no adverse effects due to vector integration and expression.