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Isolation, growth and identification of colony-forming cells with erythroid, myeloid, dendritic cell and NK-cell potential from human fetal liver.

Muench MO, Suskind DL, Bárcena A - Biol Proced Online (2002)

Bottom Line: We describe a method for the isolation from human fetal liver of highly purified candidate HSCs and progenitors based on the phenotypes CD38(-)CD34(++) and CD38(+)CD34(++), respectively.We also describe a method for the growth of colony-forming cells (CFCs) from these cell populations, under defined culture conditions, that supports the differentiation of erythroid, CD14/CD15(+) myeloid, CD1a(+) dendritic cell and CD56(+) NK cell lineages.Flow cytometric analyses of individual colonies demonstrate that CFCs with erythroid, myeloid and lymphoid potential are distributed among both the CD38(-) and CD38(+) populations of CD34(++) progenitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Laboratory Medicine, University of California at San Francisco. 3rd & Parnassus Ave., Room U-440; San Francisco, CA 94143-0793. muench@itsa.ucsf.edu

ABSTRACT
The study of hematopoietic stem cells (HSCs) and the process by which they differentiate into committed progenitors has been hampered by the lack of in vitro clonal assays that can support erythroid, myeloid and lymphoid differentiation. We describe a method for the isolation from human fetal liver of highly purified candidate HSCs and progenitors based on the phenotypes CD38(-)CD34(++) and CD38(+)CD34(++), respectively. We also describe a method for the growth of colony-forming cells (CFCs) from these cell populations, under defined culture conditions, that supports the differentiation of erythroid, CD14/CD15(+) myeloid, CD1a(+) dendritic cell and CD56(+) NK cell lineages. Flow cytometric analyses of individual colonies demonstrate that CFCs with erythroid, myeloid and lymphoid potential are distributed among both the CD38(-) and CD38(+) populations of CD34(++) progenitors.

No MeSH data available.


Flow cytometric analysis of myelocyte and DC content in individual colonies.  Results of five colonies differing in their lineage composition are shown.  Low side scatter cells are shown in blue and high side scatter cells are shown in red as indicated in A.  A colony with myeloid cells but no CD1a+ DCs is shown in B.  C-E show colonies with varying frequencies of CD1a+ and CD14/CD15+ cells.  F is an example of a colony primarily composed of CD1a+ DCs. All colonies shown were derived from CD38- progenitors.
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Figure 4: Flow cytometric analysis of myelocyte and DC content in individual colonies. Results of five colonies differing in their lineage composition are shown. Low side scatter cells are shown in blue and high side scatter cells are shown in red as indicated in A. A colony with myeloid cells but no CD1a+ DCs is shown in B. C-E show colonies with varying frequencies of CD1a+ and CD14/CD15+ cells. F is an example of a colony primarily composed of CD1a+ DCs. All colonies shown were derived from CD38- progenitors.

Mentions: CD1a+ cells were found in approximately three-quarters of all colonies grown from CD38- and CD38+ progenitors. Examples of CD1a expression on cells isolated from representative colonies are shown in Fig. 4. These colonies were stained with anti-CD56-FITC, anti-CD1a-PE, anti-CD14-APC, anti-CD15-APC and PI, enabling us to distinguish non-DC myeloid cells from DCs. The stained cells were analyzed on a two-laser FACSCalibur flow cytometer. Colonies containing CD14/CD15+ myeloid cells, but no DCs, were observed such as shown in Fig. 4B. Varying levels of myeloid and DCs were also observed as exampled by Fig. 4C-E. Note that the expression of CD14 on CD1a+ cells was variable in these colonies (CD15 is not expressed by the high side-scatter cells under these culture conditions). Fig. 4F is an example of a colony containing primarily DCs and no myeloid cells. Thus, using the techniques described the clonal analysis of CFCs capable of erythroid, myeloid, DC and NK cell differentiation is feasible.


Isolation, growth and identification of colony-forming cells with erythroid, myeloid, dendritic cell and NK-cell potential from human fetal liver.

Muench MO, Suskind DL, Bárcena A - Biol Proced Online (2002)

Flow cytometric analysis of myelocyte and DC content in individual colonies.  Results of five colonies differing in their lineage composition are shown.  Low side scatter cells are shown in blue and high side scatter cells are shown in red as indicated in A.  A colony with myeloid cells but no CD1a+ DCs is shown in B.  C-E show colonies with varying frequencies of CD1a+ and CD14/CD15+ cells.  F is an example of a colony primarily composed of CD1a+ DCs. All colonies shown were derived from CD38- progenitors.
© Copyright Policy
Related In: Results  -  Collection

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Figure 4: Flow cytometric analysis of myelocyte and DC content in individual colonies. Results of five colonies differing in their lineage composition are shown. Low side scatter cells are shown in blue and high side scatter cells are shown in red as indicated in A. A colony with myeloid cells but no CD1a+ DCs is shown in B. C-E show colonies with varying frequencies of CD1a+ and CD14/CD15+ cells. F is an example of a colony primarily composed of CD1a+ DCs. All colonies shown were derived from CD38- progenitors.
Mentions: CD1a+ cells were found in approximately three-quarters of all colonies grown from CD38- and CD38+ progenitors. Examples of CD1a expression on cells isolated from representative colonies are shown in Fig. 4. These colonies were stained with anti-CD56-FITC, anti-CD1a-PE, anti-CD14-APC, anti-CD15-APC and PI, enabling us to distinguish non-DC myeloid cells from DCs. The stained cells were analyzed on a two-laser FACSCalibur flow cytometer. Colonies containing CD14/CD15+ myeloid cells, but no DCs, were observed such as shown in Fig. 4B. Varying levels of myeloid and DCs were also observed as exampled by Fig. 4C-E. Note that the expression of CD14 on CD1a+ cells was variable in these colonies (CD15 is not expressed by the high side-scatter cells under these culture conditions). Fig. 4F is an example of a colony containing primarily DCs and no myeloid cells. Thus, using the techniques described the clonal analysis of CFCs capable of erythroid, myeloid, DC and NK cell differentiation is feasible.

Bottom Line: We describe a method for the isolation from human fetal liver of highly purified candidate HSCs and progenitors based on the phenotypes CD38(-)CD34(++) and CD38(+)CD34(++), respectively.We also describe a method for the growth of colony-forming cells (CFCs) from these cell populations, under defined culture conditions, that supports the differentiation of erythroid, CD14/CD15(+) myeloid, CD1a(+) dendritic cell and CD56(+) NK cell lineages.Flow cytometric analyses of individual colonies demonstrate that CFCs with erythroid, myeloid and lymphoid potential are distributed among both the CD38(-) and CD38(+) populations of CD34(++) progenitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Laboratory Medicine, University of California at San Francisco. 3rd & Parnassus Ave., Room U-440; San Francisco, CA 94143-0793. muench@itsa.ucsf.edu

ABSTRACT
The study of hematopoietic stem cells (HSCs) and the process by which they differentiate into committed progenitors has been hampered by the lack of in vitro clonal assays that can support erythroid, myeloid and lymphoid differentiation. We describe a method for the isolation from human fetal liver of highly purified candidate HSCs and progenitors based on the phenotypes CD38(-)CD34(++) and CD38(+)CD34(++), respectively. We also describe a method for the growth of colony-forming cells (CFCs) from these cell populations, under defined culture conditions, that supports the differentiation of erythroid, CD14/CD15(+) myeloid, CD1a(+) dendritic cell and CD56(+) NK cell lineages. Flow cytometric analyses of individual colonies demonstrate that CFCs with erythroid, myeloid and lymphoid potential are distributed among both the CD38(-) and CD38(+) populations of CD34(++) progenitors.

No MeSH data available.