Clonal Heterogeneity in Growth Kinetics of CD34+CD38– Human Cord Blood Cells In Vitro Is Correlated with Gene Expression Pattern and Telomer
http://www.100md.com
《干细胞学杂志》
a Departments of Hematology/Oncology and
c Obstetrics/Gynecology, University Medical Center, Tübingen, Germany;
b Department of Hematology/Oncology, University Medical Center, Frankfurt/Main, Germany
Key Words. CD34+ ? Telomere ? Telomerase ? Cell-cycle heterogeneity ? Gene expression ? Cord blood ? Hematopoietic progentitor cells
Correspondence: Tim H. Brümmendorf, M.D., Department of Oncology and Hematology, University Hospital Hamburg-Eppendorf, Martinistra?e 52, 20246 Hamburg, Germany. Telephone: 49-40-42803-3552; Fax: 49-40-42803-3563; e-mail: t.bruemmendorf@uke.uni-hamburg.de
ABSTRACT
Hematopoietic stem cells (HSCs) are characterized by their ability to differentiate into all hematopoietic lineages while retaining their capacity for self-renewal as well as by an extensive proliferation capacity that decreases during ontogeny . The heterogeneous composition of the human HSC compartment is poorly understood due to the lack of experimental tools that allow the characterization of the developmental program of individual stem cells . According to our current knowledge, the HSC pool may be separated into several distinct subpopulations based on both surface marker expression and retrospective identification of HSCs, using in vivo and in vitro stem cell assays. The CD34 antigen has become the major positive marker for human hematopoietic stem and progenitor cells . In human fetal liver, umbilical cord blood (CB), and bone marrow, 0.5%–5% of hematopoietic cells express CD34 , and cells with this phenotype harbor virtually all in vitro clonogenic potential . However, the pool of human hematopoietic cells defined by CD34 expression is heterogeneous. A small fraction of CD34+ cells (1%–10%) that does not express mature lineage markers (or CD38) contains cells with in vitro bilineage, lymphoid (B/NK), and myeloid differentiation potential . Furthermore, CD34+CD38– cells and not CD34+CD38+ cells are highly enriched for long-term culture-initiating cells and contain severe combined immunodeficiency (SCID)-hu–repopulating as well as nonobese diabetic SCID–repopulating cells .
In previous studies, we analyzed the functional heterogeneity of single-sorted CD34+CD38– cells from human fetal liver in growth factor–supplemented serum-free medium in vitro . The number of cells in primary cultures varied widely after 6–10 days. When CD34+CD38– cells derived from slowly growing clones were recloned, the number of cells in their respective sub-clones varied widely again. These results were indicative of a symmetric cell divisions in primitive hematopoietic cells in which proliferative potential and cell-cycle properties are unevenly distributed among daughter cells .
The aim of the present study was the characterization of the clonal composition of the CD34+CD38– compartment from an ontogenetically later but clinically more relevant HSC source, i.e., human umbilical CB. Slow growth kinetics in vitro were correlated with telomere length and gene expression profiling to potentially identify new parameters that might help to define immature subpopulations among CD34+CD38– candidate HSCs.
MATERIALS AND METHODS
Categorization of Primary Cultures Derived From Single CD34+CD38– Umbilical CB Cells
Five hundred ninety-five single CD34+CD38– CB candidate stem cells from four independent human CB samples were sorted in individual wells of round-bottomed tissue plates containing serum-free medium supplemented with human growth factors. Within 24 hours after sorting, a single viable cell was detected in 352 wells (plating efficiency, 59%) with the use of an inverted microscope. The cell number was analyzed in regular (mostly 3-day) time intervals. Two hundred ninety-six of the single-sorted cells proliferated to more than 50 cells per well (cloning efficiency, 50%). More than 11% of the single-sorted cells reached a clone size of > 1 x 105 cells and were transferred into 1-ml cultures of 24-well plates. A total of 27 transferred clones (5%) achieved a sufficient cell number (minimum 2 x 105 cells/well) for telomere-length analysis by flow FISH. Based on the time span it took the individual clone to reach 1 x 105 cells, the transferred clones were categorized into two groups. The median duration to expand to 1 x 105 cells was 36 days. Accordingly, clones that required 36 days were classified as fast, whereas the remaining ones were classified as slowly growing clones.
Growth Kinetics of Highly Proliferative CB Clones
The growth kinetics of 27 transferred clones that achieved a clone size of 1 x 105 cells between days 30 and 57 is graphed in Figure 3. After individual clones were transferred into 24-well plates, the cell number was not counted any more until the clones were harvested for flow FISH analysis. According to the median time span it took individual clones to reach 1 x 105 cells (i.e., 36 days), 20 of the transferred clones were classified as fast (36 days) and 7 as slowly growing clones (>36 days). After 12–15 days in culture, the number of cells per well varied over a wide range, indicating extensive heterogeneity among CB CD34+CD38– cells. On day 27, rapidly growing clones yielded an average cell number (mean ± standard error of mean ) of 48,000 ± 6,000, compared with 27,000 ± 10,000 found in slowly growing clones.
Figure 3. Clonal heterogeneity in single-sorted CD34+CD38– human umbilical cord blood cells in growth factor–supplemented serum-free medium. At the indicated time intervals, the number of cells (mean ± standard error ) in each well was scored under an inverted microscope. Here, clones that required less or equal as compared with more than 36 days to reach the level of 105 cells per well were classified as fast-growing (n = 20) or slowly growing (n = 7), respectively. The plot shows the growth kinetics from the day when single cells were sorted until the first time point, when individual clones were transferred into 24-well plates.
Telomere-Length Measurements of CB Clones
We could observe a substantial heterogeneity in telomere length between the 27 highly proliferative clones, ranging from 9 to 23 kMESF. A significant negative linear correlation between growth kinetics and telomere length was observed (not shown). Slowly growing clones (n = 7), which have previously been characterized by a high proliferative potential , showed significantly longer telomere length compared with the group of fast-growing clones (n = 20). The mean difference in telomere fluorescence between fast-growing (mean ± SEM, 13.7 ± 0.6 kMESF) and slowly growing (20.1 ± 0.6 kMESF) clones amounted to 6.4 kMESF (Fig. 4; p < .001), which is equivalent to approximately 3.2 kb.
Figure 4. Telomere length analysis by flow fluorescence in situ hybridization in clones (n = 27) derived from CD34+CD38– human umbilical cord blood cells cultured in serum-free medium supplemented with stem cell factor, Flt-3, interleukin-3, interleukin-6, thrombopoietin, and G-CSF. Mean telomere length (black bar) was expressed for the category of slowly growing clones compared with the category of the fast-growing clones. Slowly growing clones (n = 7, including three single values) showed significantly (p < .001) longer telomeres compared with the fast-growing clones (n = 20, including seven single values).
Methylcellulose Progenitor Assay for Colony-Forming Units
To investigate the colony-forming capacity in fast compared with slowly growing clones, a methylcellulose colony assay was used. A total of 50 clones from three human umbilical CB specimens achieved a clone size of 1 x 104 cells per well between days 12 and 28. According to the median time span it took individual clones to reach 1 x 104 cells (i.e., 18 days), 25 of the clones were classified as fast-growing or slowly growing clones, respectively. When achieving the target cell number of 1 x 104 cells per well (Fig. 1B), respective cells were directly put in a methylcellulose assay. The average number of CFU-GM and BFU-E colonies of each clone is graphed in Figure 5. Colony-forming capacity was detected in 21 of 25 clones from the fast-growing category (84%) compared with 20 of 25 clones from the slowly growing category (80%). In the category of fast-growing clones, a total of 753 colonies were detected. Of these, 17 colonies (2.3%) were defined as erythroid and 736 colonies (97.7%) were defined as myeloid colonies. In the fraction of slowly growing clones, a total of 751 colonies were counted, 25 (3.3%) of which were classified as erythroid and 726 (96.7%) of which were defined as myeloid colonies. No significant difference in the median number of CFU-GM and BFU-E between fast-growing versus slowly growing clones was observed.
Figure 5. Methylcellulose assay illustrating the median (black bar) of lineage-committed progenitor cell capacity in the category of slowly growing clones (n = 25) compared with the category of fast-growing clones (n = 25) in vitro.
Gene Expression Pattern in Clones Derived from Single CD34+CD38– Human CB Cells
We analyzed differential gene expression in a total of 134 clones derived from 1440 single-sorted CD34+CD38– cells in two independent experiments with three CB samples each. These 134 clones reached a clone size 1 x 104 per well between 12 and 30 days in culture. The median time to expand to 1 x 104 cells was 18 days in both independent experiments. Accordingly, clones that required less time than the median were classified as fast (a total of 79 clones), whereas the remaining clones were classified as slowly growing clones (a total of 55 clones). Clones of each category were pooled for gene expression analysis by oligonucleotide microarrays. For a first analysis, genes that are differentially expressed in fast-growing clones compared with slowly growing clones were required to pass the restrictions in all (three of three) paired samples analyzed. A twofold upregulation of 115 genes was detected in slowly growing clones (Table 1), whereas 43 genes showed a downregulation compared with fast-growing clones (Table 2). In further analyses, differentially expressed genes that were upregulated were required to have the Affymetrixcall present, and those that were downregulated were required to have the Affymetrixcall absent in all of the samples. This resulted in a selection of 10 genes that could be used to clearly separate fast-growing clones from slowly growing clones by hierarchical cluster analysis (Fig. 6). Among the five genes overexpressed in slowly growing clones, three seemed to be of particular interest. Pax4 (paired box gene 4) is involved in embryonal development, CD61 (platelet glycoprotein IIIa) is involved in megakaryopoesis, and ETB (endothelin receptor type B) is involved in cell signaling.
Table 1. Genes upregulated in slowly growing clones compared with fast-growing clones derived from individual CD34+CD38– cells from human umbilical cord blood
Table 2. Genes downregulated in slowly growing clones compared with fast-growing clones derived from individual CD34+CD38– cells from human umbilical cord blood
Figure 6. Identification of clusters of gene expression in clones derived from CD34+CD38– side scatter from human umbilical cord blood. Increasing the level of statistical restrictions resulted in the selection of 10 genes (vertical list: gene and accession number) that subsequently could be used to clearly separate fast from slowly growing clones by hierarchical clustering with Spearman’s confidence correlation. Horizontal list is each of the samples. Vertical list displays the 10 genes. Color code: Blue, low expression; Red, high expression. The intensity of the color reflects the trust of the expression data.
Genes that are differentially expressed in the two categories and passed the restrictions were classified as either hematopoiesis- or nonhematopoiesis-affiliated genes according to their tissue-specific expression or functions (Fig. 7). Most of the non-hematopoiesis-affiliated genes are present in the group of slowly growing clones (21%) compared with fast-growing clones (7%). Furthermore, genes involved in embryonal development and early hematopoiesis are predominantly found in the slowly growing fraction. Representative candidate genes are Pax4, Meis (mouse) homologue 2, Ang-1, and SCL/tal-1 (Table 1).
Figure 7. Gene expression profiles were analyzed in clones (n = 134) derived from single-sorted CD34+CD38– human umbilical cord blood cells. Categorization of informative sequences by function (A) or by tissue (B) that were upregulated in the category of slowly growing clones (n = 55) compared with the category of fast-growing clones (n = 79) derived from single-sorted CD34+CD38– cells from human umbilical cord blood (n = 3).
DISCUSSION
This study was supported by the Sonderforschungsbereich 510 (Teilprojekt A6) of the Deutsche Forschungsgemeinschaft (Bonn, Germany). We would like to thank Alexandra Wahl and Anke Marxer for excellent technical assistance and Dr. Martin Eichner (Department of Medical Biometry, Tübingen University, Tübingen, Germany) for help with statistical analysis of the data. Furthermore, we would like to thank Andreas M. Boehmler for critical reading of the manuscript.
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c Obstetrics/Gynecology, University Medical Center, Tübingen, Germany;
b Department of Hematology/Oncology, University Medical Center, Frankfurt/Main, Germany
Key Words. CD34+ ? Telomere ? Telomerase ? Cell-cycle heterogeneity ? Gene expression ? Cord blood ? Hematopoietic progentitor cells
Correspondence: Tim H. Brümmendorf, M.D., Department of Oncology and Hematology, University Hospital Hamburg-Eppendorf, Martinistra?e 52, 20246 Hamburg, Germany. Telephone: 49-40-42803-3552; Fax: 49-40-42803-3563; e-mail: t.bruemmendorf@uke.uni-hamburg.de
ABSTRACT
Hematopoietic stem cells (HSCs) are characterized by their ability to differentiate into all hematopoietic lineages while retaining their capacity for self-renewal as well as by an extensive proliferation capacity that decreases during ontogeny . The heterogeneous composition of the human HSC compartment is poorly understood due to the lack of experimental tools that allow the characterization of the developmental program of individual stem cells . According to our current knowledge, the HSC pool may be separated into several distinct subpopulations based on both surface marker expression and retrospective identification of HSCs, using in vivo and in vitro stem cell assays. The CD34 antigen has become the major positive marker for human hematopoietic stem and progenitor cells . In human fetal liver, umbilical cord blood (CB), and bone marrow, 0.5%–5% of hematopoietic cells express CD34 , and cells with this phenotype harbor virtually all in vitro clonogenic potential . However, the pool of human hematopoietic cells defined by CD34 expression is heterogeneous. A small fraction of CD34+ cells (1%–10%) that does not express mature lineage markers (or CD38) contains cells with in vitro bilineage, lymphoid (B/NK), and myeloid differentiation potential . Furthermore, CD34+CD38– cells and not CD34+CD38+ cells are highly enriched for long-term culture-initiating cells and contain severe combined immunodeficiency (SCID)-hu–repopulating as well as nonobese diabetic SCID–repopulating cells .
In previous studies, we analyzed the functional heterogeneity of single-sorted CD34+CD38– cells from human fetal liver in growth factor–supplemented serum-free medium in vitro . The number of cells in primary cultures varied widely after 6–10 days. When CD34+CD38– cells derived from slowly growing clones were recloned, the number of cells in their respective sub-clones varied widely again. These results were indicative of a symmetric cell divisions in primitive hematopoietic cells in which proliferative potential and cell-cycle properties are unevenly distributed among daughter cells .
The aim of the present study was the characterization of the clonal composition of the CD34+CD38– compartment from an ontogenetically later but clinically more relevant HSC source, i.e., human umbilical CB. Slow growth kinetics in vitro were correlated with telomere length and gene expression profiling to potentially identify new parameters that might help to define immature subpopulations among CD34+CD38– candidate HSCs.
MATERIALS AND METHODS
Categorization of Primary Cultures Derived From Single CD34+CD38– Umbilical CB Cells
Five hundred ninety-five single CD34+CD38– CB candidate stem cells from four independent human CB samples were sorted in individual wells of round-bottomed tissue plates containing serum-free medium supplemented with human growth factors. Within 24 hours after sorting, a single viable cell was detected in 352 wells (plating efficiency, 59%) with the use of an inverted microscope. The cell number was analyzed in regular (mostly 3-day) time intervals. Two hundred ninety-six of the single-sorted cells proliferated to more than 50 cells per well (cloning efficiency, 50%). More than 11% of the single-sorted cells reached a clone size of > 1 x 105 cells and were transferred into 1-ml cultures of 24-well plates. A total of 27 transferred clones (5%) achieved a sufficient cell number (minimum 2 x 105 cells/well) for telomere-length analysis by flow FISH. Based on the time span it took the individual clone to reach 1 x 105 cells, the transferred clones were categorized into two groups. The median duration to expand to 1 x 105 cells was 36 days. Accordingly, clones that required 36 days were classified as fast, whereas the remaining ones were classified as slowly growing clones.
Growth Kinetics of Highly Proliferative CB Clones
The growth kinetics of 27 transferred clones that achieved a clone size of 1 x 105 cells between days 30 and 57 is graphed in Figure 3. After individual clones were transferred into 24-well plates, the cell number was not counted any more until the clones were harvested for flow FISH analysis. According to the median time span it took individual clones to reach 1 x 105 cells (i.e., 36 days), 20 of the transferred clones were classified as fast (36 days) and 7 as slowly growing clones (>36 days). After 12–15 days in culture, the number of cells per well varied over a wide range, indicating extensive heterogeneity among CB CD34+CD38– cells. On day 27, rapidly growing clones yielded an average cell number (mean ± standard error of mean ) of 48,000 ± 6,000, compared with 27,000 ± 10,000 found in slowly growing clones.
Figure 3. Clonal heterogeneity in single-sorted CD34+CD38– human umbilical cord blood cells in growth factor–supplemented serum-free medium. At the indicated time intervals, the number of cells (mean ± standard error ) in each well was scored under an inverted microscope. Here, clones that required less or equal as compared with more than 36 days to reach the level of 105 cells per well were classified as fast-growing (n = 20) or slowly growing (n = 7), respectively. The plot shows the growth kinetics from the day when single cells were sorted until the first time point, when individual clones were transferred into 24-well plates.
Telomere-Length Measurements of CB Clones
We could observe a substantial heterogeneity in telomere length between the 27 highly proliferative clones, ranging from 9 to 23 kMESF. A significant negative linear correlation between growth kinetics and telomere length was observed (not shown). Slowly growing clones (n = 7), which have previously been characterized by a high proliferative potential , showed significantly longer telomere length compared with the group of fast-growing clones (n = 20). The mean difference in telomere fluorescence between fast-growing (mean ± SEM, 13.7 ± 0.6 kMESF) and slowly growing (20.1 ± 0.6 kMESF) clones amounted to 6.4 kMESF (Fig. 4; p < .001), which is equivalent to approximately 3.2 kb.
Figure 4. Telomere length analysis by flow fluorescence in situ hybridization in clones (n = 27) derived from CD34+CD38– human umbilical cord blood cells cultured in serum-free medium supplemented with stem cell factor, Flt-3, interleukin-3, interleukin-6, thrombopoietin, and G-CSF. Mean telomere length (black bar) was expressed for the category of slowly growing clones compared with the category of the fast-growing clones. Slowly growing clones (n = 7, including three single values) showed significantly (p < .001) longer telomeres compared with the fast-growing clones (n = 20, including seven single values).
Methylcellulose Progenitor Assay for Colony-Forming Units
To investigate the colony-forming capacity in fast compared with slowly growing clones, a methylcellulose colony assay was used. A total of 50 clones from three human umbilical CB specimens achieved a clone size of 1 x 104 cells per well between days 12 and 28. According to the median time span it took individual clones to reach 1 x 104 cells (i.e., 18 days), 25 of the clones were classified as fast-growing or slowly growing clones, respectively. When achieving the target cell number of 1 x 104 cells per well (Fig. 1B), respective cells were directly put in a methylcellulose assay. The average number of CFU-GM and BFU-E colonies of each clone is graphed in Figure 5. Colony-forming capacity was detected in 21 of 25 clones from the fast-growing category (84%) compared with 20 of 25 clones from the slowly growing category (80%). In the category of fast-growing clones, a total of 753 colonies were detected. Of these, 17 colonies (2.3%) were defined as erythroid and 736 colonies (97.7%) were defined as myeloid colonies. In the fraction of slowly growing clones, a total of 751 colonies were counted, 25 (3.3%) of which were classified as erythroid and 726 (96.7%) of which were defined as myeloid colonies. No significant difference in the median number of CFU-GM and BFU-E between fast-growing versus slowly growing clones was observed.
Figure 5. Methylcellulose assay illustrating the median (black bar) of lineage-committed progenitor cell capacity in the category of slowly growing clones (n = 25) compared with the category of fast-growing clones (n = 25) in vitro.
Gene Expression Pattern in Clones Derived from Single CD34+CD38– Human CB Cells
We analyzed differential gene expression in a total of 134 clones derived from 1440 single-sorted CD34+CD38– cells in two independent experiments with three CB samples each. These 134 clones reached a clone size 1 x 104 per well between 12 and 30 days in culture. The median time to expand to 1 x 104 cells was 18 days in both independent experiments. Accordingly, clones that required less time than the median were classified as fast (a total of 79 clones), whereas the remaining clones were classified as slowly growing clones (a total of 55 clones). Clones of each category were pooled for gene expression analysis by oligonucleotide microarrays. For a first analysis, genes that are differentially expressed in fast-growing clones compared with slowly growing clones were required to pass the restrictions in all (three of three) paired samples analyzed. A twofold upregulation of 115 genes was detected in slowly growing clones (Table 1), whereas 43 genes showed a downregulation compared with fast-growing clones (Table 2). In further analyses, differentially expressed genes that were upregulated were required to have the Affymetrixcall present, and those that were downregulated were required to have the Affymetrixcall absent in all of the samples. This resulted in a selection of 10 genes that could be used to clearly separate fast-growing clones from slowly growing clones by hierarchical cluster analysis (Fig. 6). Among the five genes overexpressed in slowly growing clones, three seemed to be of particular interest. Pax4 (paired box gene 4) is involved in embryonal development, CD61 (platelet glycoprotein IIIa) is involved in megakaryopoesis, and ETB (endothelin receptor type B) is involved in cell signaling.
Table 1. Genes upregulated in slowly growing clones compared with fast-growing clones derived from individual CD34+CD38– cells from human umbilical cord blood
Table 2. Genes downregulated in slowly growing clones compared with fast-growing clones derived from individual CD34+CD38– cells from human umbilical cord blood
Figure 6. Identification of clusters of gene expression in clones derived from CD34+CD38– side scatter from human umbilical cord blood. Increasing the level of statistical restrictions resulted in the selection of 10 genes (vertical list: gene and accession number) that subsequently could be used to clearly separate fast from slowly growing clones by hierarchical clustering with Spearman’s confidence correlation. Horizontal list is each of the samples. Vertical list displays the 10 genes. Color code: Blue, low expression; Red, high expression. The intensity of the color reflects the trust of the expression data.
Genes that are differentially expressed in the two categories and passed the restrictions were classified as either hematopoiesis- or nonhematopoiesis-affiliated genes according to their tissue-specific expression or functions (Fig. 7). Most of the non-hematopoiesis-affiliated genes are present in the group of slowly growing clones (21%) compared with fast-growing clones (7%). Furthermore, genes involved in embryonal development and early hematopoiesis are predominantly found in the slowly growing fraction. Representative candidate genes are Pax4, Meis (mouse) homologue 2, Ang-1, and SCL/tal-1 (Table 1).
Figure 7. Gene expression profiles were analyzed in clones (n = 134) derived from single-sorted CD34+CD38– human umbilical cord blood cells. Categorization of informative sequences by function (A) or by tissue (B) that were upregulated in the category of slowly growing clones (n = 55) compared with the category of fast-growing clones (n = 79) derived from single-sorted CD34+CD38– cells from human umbilical cord blood (n = 3).
DISCUSSION
This study was supported by the Sonderforschungsbereich 510 (Teilprojekt A6) of the Deutsche Forschungsgemeinschaft (Bonn, Germany). We would like to thank Alexandra Wahl and Anke Marxer for excellent technical assistance and Dr. Martin Eichner (Department of Medical Biometry, Tübingen University, Tübingen, Germany) for help with statistical analysis of the data. Furthermore, we would like to thank Andreas M. Boehmler for critical reading of the manuscript.
REFERENCES
Till JE, McCulloch EA. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 1961;14:213–222.
Lansdorp PM, Dragowska W, Mayani H. Ontogeny-related changes in proliferative potential of human hematopoietic cells. J Exp Med 1993;178:787–791.
Guenechea G, Gan OI, Dorrell C et al. Distinct classes of human stem cells that differ in proliferative and self-renewal potential. Nat Immunol 2001;2:75–82.
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