Use of Multicolor Flow Cytometry for Isolation of Specific Cell Populations Deriving from Differentiated Human Embryonic Stem Cells

2013 ◽  
pp. 191-203
Author(s):  
Isabella Mengarelli ◽  
Andrew Fryga ◽  
Tiziano Barberi
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Cell Populations ◽  
Specific Cell ◽  
Multicolor Flow Cytometry

The derivation and potential use of human embryonic stem cells

2001 ◽  
Vol 13 (8) ◽  
pp. 523 ◽  
Author(s):  
Alan O. Trounson
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Regenerative Medicine ◽  
Human Embryonic Stem Cells ◽  
High Efficiency ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Types ◽  
Specific Cell

Human embryonic stem cells lines can be derived from human blastocysts at high efficiency (>50%) by immunosurgical isolation of the inner cell mass and culture on embryonic fibroblast cell lines. These cells will spontaneously differentiate into all the primary embryonic lineages in vitro and in vivo, but they are unable to form an integrated embryo or body plan by themselves or when combined with trophectoderm cells. They may be directed into a number of specific cell types and this enrichment process requires specific growth factors, cell-surface molecules, matrix molecules and secreted products of other cell types. Embryonic stem (ES) cells are immortal and represent a major potential for cell therapies for regenerative medicine. Their use in transplantation may depend on the formation of a large bank of suitable human leucocyte antigen (HLA) types or the genetic erasure of their HLA expression. Successful transplantation may also require induction of tolerance in recipients and ongoing immune suppression. Although it is possible to customize ES cells by therapeutic cloning or cytoplasmic transfer, it would appear unlikely that these strategies will be used extensively for producing ES cells compatible for transplantation. Embryonic stem cell research may deliver a new pathway for regenerative medicine.

Differentiation of Human Embryonic Stem Cells into Mesenchymal Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1389-1389
Author(s):  
Emmanuel N. Olivier ◽  
Anne C. Rybicki ◽  
Eric E. Bouhassira
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Therapeutic Potential ◽  
Embryonic Stem ◽  
Specific Cell ◽  
Alizarin Red ◽  
Morphological Criteria

Abstract Mesenchymal stem cells (MSC) are multipotent progenitors that contribute to the formation of many connective tissues including fat, bone, cartilage and muscle. Because of this great versatility MSCs have a large therapeutic potential particularly in the areas of cell therapy and regenerative and reconstructive medicine. MSCs are rare cells that can be isolated from tissues such as bone marrow, cartilage and muscles. Since no unique marker characteristic of MSCs has been identified, investigators have relied on a series of functional and morphological criteria to identify them. These criteria include growth on plastic, resistance to trypsin, presence of specific cell surface antigens and potential to differentiate into adipocytes, chondrocytes and osteoblasts.We report here a method to reproducibly differentiate human embryonic stem cells (hESCs) into MSCs by pre-differentiating for 8 days hESCs growing on MEF, mechanically dissociating the differentiated colonies, replating the differentiated colonies in DMEM, 10% FBS and 7.5% CO2 for 4 to 8 weeks until a thick multi-layer epithelium-like sheet of cells develop; and dissociating these multi-layer structures with a combination of proteases. The cells obtained with this procedure are morphologically similar to MSCs, are contact-inhibited, can be grown in culture for about 20–25 passages and have a gene expression profile, as determined by cDNA micro-array, similar to published profiles of mesenchymal stem cells. Immuno-phenotyping of these hESC-derived MSCs revealed a immuno-profile similar to bone marrow MSCs (negative for CD34, CD45 and positive for CD13, CD44, CD71, CD73, CD105, CD166, HLA ABC, SSEA4 and TRA1-85). Functional differentiation experiments revealed that hESC-derived MSCs can be differentiated into osteocytes and adipocytes as demonstrated by staining with alizarin red and oil red O. Finally, we have also shown that hESC-derived MSCs can support the growth of undifferentiated hESCs cells and of CD34+ hematopoietic cells.The ability to produce MSCs from hESCs should prove useful to produce large amount of genetically identical and genetically modifiable MSCS that can be used to study the biology of MSCs and for therapeutic applications.

scholarly journals Multiparameter flow cytometry for the characterization of human embryonic stem cells

2012 ◽  
Vol 35 (1) ◽  
pp. 55-65 ◽  
Author(s):  
Kathryn Brosnan ◽  
Andrew Want ◽  
Karen Coopman ◽  
Christopher J. Hewitt
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Multiparameter Flow Cytometry

Potential benefits of cell cloning for human medicine

1998 ◽  
Vol 10 (1) ◽  
pp. 121 ◽  
Author(s):  
A. Trounson ◽  
M. Pera
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Somatic Cell ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Specific Cell ◽  
Somatic Cell Nucleus ◽  
Potential Benefits

The successful cloning of a mammal from an adult somatic cell nucleus opens new avenues for major advances in reproductive medicine, biotechnology and cellular-based transplantation therapies for degenerative diseases. At the same time, this breakthrough has generated much heated discussion concerning the ethics of cloning. Twinning is a form of cloning, and there are instances in clinical assisted reproduction in which the deliberate formation of twins by embryo dissection would seem ethically acceptable. Nuclear transfer technology might facilitate the derivation of human embryonic stem cells, capable of differentiation into a wide variety of somatic cell lineages. Directed differentiation of human embryonic stem cells into specific cell types in vitro could provide a universal source of cells for transplantation therapy. The potential benefits of therapeutics based on cloning technologies are considerable, and hasty legislation to ban all such procedures could block progress in critical arenas of biomedical research

scholarly journals αVβ5 and CD44 Are Oxygen-Regulated Human Embryonic Stem Cell Attachment Factors

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Deepak Kumar ◽  
Saniya Gupta ◽  
Ying Yang ◽  
Nicholas R. Forsyth
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Human Embryonic Stem Cell ◽  
Cultured Cells ◽  
Cell Attachment ◽  
Embryonic Stem ◽  
Cd44 Expression

Human embryonic stem cells (hESCs) have great potential for clinical therapeutic use. However, relatively little is known of the mechanisms which dictate their specificity of adhesion to substrates through adhesion proteins including integrins. Previous observations demonstrated enhanced clonogenicity in reduced oxygen culture systems. Here, we demonstrated via antibody blocking experiments thatαVβ5 andα6 significantly promoted hESC attachment in 2% O2only, whereas blockage of CD44 inhibited cell attachment in 21% O2alone. Immunofluorescence confirmed expression ofαVβ5 and CD44 in both 2% O2and 21% O2cultured hESCs while flow cytometry revealed significantly higherαVβ5 expression in 2% O2versus 21% O2cultured hESCs and higher CD44 expression in 21% O2versus 2% O2cultured hESCs. Adhered hESCs following blockage ofαVβ5 in 2% O2displayed a reduction in nuclear colocalisation of Oct-4 and Nanog with little effect observed in 21% O2. Blockage of CD44 had the converse effect with dramatic reductions in nuclear colocalisation of Oct-4 and Nanog in 21% O2cultured hESC which retained adherence, but not in 2% O2cultured cells. Identification of oxygen-dependent substrate attachment mechanisms in hESCs has the potential to play a role in the development of novel substrates to improve hESC attachment and culture.

Human Embryonic Stem Cells Differentiate into Functional Natural Killer Cells with the Capacity To Mediate Anti-Tumor Activity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 763-763
Author(s):  
Dan S. Kaufman ◽  
Petter S. Woll ◽  
Colin H. Martin ◽  
Jeffrey S. Miller
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Nk Cells ◽  
Progenitor Cells ◽  
Human Embryonic Stem Cells ◽  
Nk Cell ◽  
Embryonic Stem ◽  
Cytolytic Activity ◽  
Cell Populations ◽  
Raji Cells

Abstract Hematopoiesis from human embryonic stem cells (hESCs) follows developmental kinetics similar to what is observed during normal human ontogeny. Myeloid, erythroid and megakaryocytic progenitors can be routinely generated from hESCs. However, little is known about the ability of hESCs to differentiate into the lymphoid lineage. Natural killer (NK) cells are important mediators of donor anti-host alloreactivity seen after allogeneic transplant for myeloid leukemias. Our studies use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. CD34+ and CD34+CD45+ hESC-derived hematopoietic progenitor cells were co-cultured with inactivated AFT024 stromal cells in medium supplemented with IL-7, IL-15, SCF and FL. Generation of NK cells was established by phenotypic and functional analysis. CD34+ umbilical cord blood (UCB) cells were utilized as a positive control. After 14 days of culture of CD34+ hESC-derived cells, more than 90% of the cells express CD45, a pan-hematopoietic cell marker, but few CD56+ cells are observed. At 21 days of culture a distinct CD56+CD45+ cell population develops (14.9%), which increases to 37.5% of cells after 28 days of culture. Similar results are observed for CD34+CD45+ hESC-derived cells, characterizing that both CD34+ and CD34+CD45+ cell populations contain hematopoietic progenitors with NK cell developmental potential. Limiting dilution analysis of hESC-derived progenitor cells demonstrates CD34+ hESC-derived cells have a low NK cell progenitor frequency. However, sorting for CD34+CD45+ hESC-derived cells significantly increased the NK cell cloning frequency (1.92% ± 1.20%) to a level comparable to the frequency observed for CD34+ UCB cells cultured in the same manner (3.57% ± 1.68%). The hESC-derived NK cells also express receptors known to regulate NK cell cytolytic activity, including killer-Ig-like receptors (KIRs), C-type lectin-like receptors (CD94 and NKG2A) and natural cytotoxicity receptors (NKp30, NKp44, and NKp46). Furthermore, hESC-derived NK cells also express CD16, an Fc-receptor typically expressed on more mature NK cells. The expression of KIRs is significantly higher for the hESC-derived NK cells compared to the UCB-derived NK cells. This may lead to future strategies to generate selective alloreactive NK cell populations for therapy. To investigate the functional properties of the hESC-derived NK cells, cytolytic activity was tested against K562 erythroleukemia cells and Raji B-lymphoblastoid cells. hESC-derived NK cells effectively killed K562 cells, with activity similar to that seen with UCB-derived NK cells. As expected, Raji cells were resistant to direct cytotoxicity by both hESC and UCB-derived NK cells. However, treatment of Raji cells with anti-CD20 antibody results in effective antibody-dependent cell-mediated cytoxicity by the hESC-derived NK cells. The hESC-derived NK cells also demonstrate ability to upregulate production of cytokines such as IFN-γ upon stimulation. Furthermore, we also find that hESC-derived progenitors also have T cell and/or B cell potential based on cells that express Ikaros, Rag1, and IL7Rα. These results demonstrate that the CD34+ and CD34+CD45+ hESC-derived cell populations contain lymphoid progenitor cells that can develop into both innate and adaptive immune cells. The ability to generate functional NK cells that can target and lyse human tumor cells via two distinct mechanisms suggests potentially novel anti-cancer therapy applications of hESCs.

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