Characterization of a Lineage-Negative Stem-Progenitor Cell Population Optimized for Ex Vivo Expansion and Enriched for LTC-IC
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《干细胞学杂志》
a King-George Laboratory, St. George’s Hospital Medical School and Kingston University, London, UK;
b School of Life Sciences, Kingston University, Kingston upon Thames, UK;
c Department of Haematology, St. George’s Hospital Medical School, London, UK
Key Words. Negative selection ? Stem cells ? LTC-IC ? CD34 ? Ex vivo expansion
Colin P. McGuckin, Ph.D., Reader, School of Life Sciences, Faculty of Science, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, United Kingdom. Telephone: 44-797-126-6764; Fax: 44-208-725-0245; e-mail: c.mcguckin@kingston.ac.uk
ABSTRACT
Since the 1980s, hematopoietic stem cell transplantation and clinical research have mainly revolved around the cell surface protein, CD34, used as a marker for positive selection of heterogeneous hematopoietic stem and progenitor cells (HSPC) . Although the true role of the CD34 molecule continues to be debated, CD34+ HSPC have been functionally defined as capable of generating progenitor-derived clones in vitro and by their potential in reconstituting the lymphomyelopoietic system in myelocompromised hosts . CD133 was reported as an alternative marker for positive selection methods targeting more primitive HSPC enriched with CD34bright cells . However, we have recently reported that although CD133+ cells had interesting ex vivo expansion potential, they still encompassed cells at various stages of differentiation .
Several studies have demonstrated that cells that lack CD34 and hematopoietic lineage markers (Lin-/CD34-) could engraft immunocompromised animal hosts and sustain long-term in vivo hematopoiesis . Controversies then rapidly appeared by other groups suggesting limited hematopoietic engraftment potential to Lin-CD34- cells when compared with Lin-CD34+ HSPC . These investigators then hypothesized that small numbers of contaminating CD34+ HSPC could account for CD34- cell engraftment to the marrow . This controversy has been partly reconciled due to papers indicating the reversibility of CD34 expression, which may vary in vivo according to variable engraftment requirements .
Much of the problem with Lin- cell populations described so far is that they are poorly characterized. Modifications in previously described isolation protocols may also account for the variability of engraftment in immunocompromised animal models . Due to the need to find an optimized population, we have developed a reproducible strategy for primitive cell harvest.
MATERIALS AND METHODS
Cell Purification and Phenotypic Characterization
The mean purity of CD133+ immunomagnetically selected cells was 94.3% ± 0.9% and represented 0.4% ± 0.04% of the original UCB MNC (n = 19). CD133+ cells coexpressed other surface markers as follows: CD34 (93.8% ± 0.9%), CD38 (62.7% ± 3.7%), CD164 (63.8% ± 6.0%), CD162 (69.3% ± 2.6%), and CXCR4 (67.7% ± 1.4%).
Lin- HSPC were characterized as a discrete cell population representing 0.1% ± 0.02% (n = 11) of original UCB MNC. The Lin- cell subset was negative for a wide range of hematopoietic lineage markers, including CD45, glycophorin-A, CD38, CD7, CD33, CD56, CD16, CD3, and CD2. A proportion of Lin- HSPC nonetheless expressed markers: A) reflecting their immaturity status , such as CD133 (7.0% ± 0.8%), CD34 (14.4% ± 3.6%), intracellular CD34 (16.2% ± 0.6%), and CD164 (16.0% ± 4.1%), or B) that were involved in HSPC migration, adhesion, and homing to the bone marrow (BM) , CXCR4 (48.6% ± 4.0%), and CD162 (96.7% ± 2.0%) (Fig. 1). Our negative isolation protocol appeared to be highly reproducible and isolated a rare primitive Lin- cell population.
Figure 1. Phenotypic characterization of Lin- cells after isolation from UCB. Lin- cells expressed higher levels of immaturity markers, such as CD133, CD34 (n = 7), intracellular CD34 (n = 2), and CD164 (n = 5), than low-density UCB MNC. Lin- cells also expressed CXCR4 (n = 3) and CD162 (n = 3), both involved in hematopoietic progenitors homing to the BM. Results are expressed as percentage of the Lin- cell population ± SE.
CD133+ Cells Demonstrated a High Proliferation Potential in Cytokine-Stimulated Liquid Culture
Over 8 weeks in liquid culture, CD133+ cells proliferated more rapidly and yielded a significantly higher viable total cell number-fold increase (FI) than Lin- HSPC under both K36EG (p < 0.01) and TPOFLK (p < 0.05) stimulations (Fig. 2). In both culture systems, MNC were baseline and exhausted by week 7 of culture. Interestingly, TPOFLK cytokine mix induced a significantly higher viable cell FI when growing CD133+ cells for 8 weeks in culture (TPOFLK FI: 77.10 ± 0.27 x 107 versus K36EG FI: 1.53 ± 0.60 x 107, p < 0.05, n = 4). A similar pattern was observed for Lin- HSPC with a higher proliferation potential under TPOFLK synergism (week 8 FI: 33.20 ± 1.75) when compared with K36EG-stimulated liquid culture in which they stopped growing by week 6 (p < 0.001, n = 4). At first analysis, when compared with Lin- cells, CD133+ had a better ability to produce large cell numbers in TPOFLK-stimulated liquid culture.
Figure 2. CD133+ cells demonstrated a high proliferation potential in cytokine-stimulated liquid culture. Ex vivo expansion of Lin- cells, CD133+ cells, and MNC over 8 weeks in liquid culture stimulated either by TPOFLK or K36EG. Over 8 weeks, CD133+ cells proliferated more rapidly and yielded significantly higher cumulative CFC counts than Lin- cells under both K36EG (p < 0.01) and TPOFLK (p < 0.05) growth factor stimulations. When compared with K36EG stimulation, TPOFLK synergism induced significantly cumulative CFC counts for both CD133+ cells and Lin- cells. MNC did not expand CFC and exhausted by week 8. Results are expressed as mean ± SE of four separate experiments.
TPOFLK-Stimulated Liquid Culture of Lin- Cells Maintains a More Primitive Population of HSPC than CD133+ Cells
The TPOFLK-stimulated liquid culture expansion system consistently maintained a higher proportion of CD133+ and Lin- cell-derived primitive colony-forming cells (CFC) when compared with the effect K36EG cytokine mix on these cells (Fig. 3) (p < 0.05, n = 4). This confirmed the value of TPOFLK synergism when expanding primitive HSPC in liquid culture.
Figure 3. TPOFLK-stimulated liquid culture of Lin- cells maintains a more primitive population of HSPC than CD133+ cells. Lin-, CD133+, and MNC were respectively grown over 8 weeks in liquid culture stimulated either by TPOFLK or K36EG. At weeks 2, 4, 6, and 8, 104 cells from each cell type (Lin-, CD133+, and MNC), in both TPOFLK and K36EG conditions, were seeded in a clonogenic assay to enumerate CFC capacity. TPOFLK synergism consistently maintained a higher proportion of CD133+ and Lin- cell-derived CFC when compared with K36EG cytokine mix stimulation (p < 0.05). In TPOFLK-stimulated expansion system, Lin- cells produced more CFC/104 seeded cells than CD133+ cells (*p < 0.05). Results are expressed as mean ± SE of four separate experiments.
After immediate selection, CD133+ cells were enriched for more CFC/104-seeded cells (485 ± 111) when compared with Lin- cells (251 ± 41) CFC and MNC (12 ± 2) (p < 0.05, n = 5). However, in TPOFLK-stimulated liquid culture, Lin- cells consistently maintained a higher proportion of CFC at weeks 4, 6, and 8 than CD133+ cells and MNC (Fig. 3). This difference was particularly significant at 6 and 8 weeks (p < 0.05). Interestingly, in TPOFLK-stimulated liquid culture, the peak in CFC maintenance/expansion from Lin- cells was observed at 6 weeks of expansion (34 ± 8 CFC/104 cells), correlating with their slow proliferation pattern. Under the same conditions, the highest CFC frequency from CD133+ cells appeared earlier at week 4 and gradually faded by week 8 (Fig. 3).
After selection, both Lin- and CD133+ cells were predominantly enriched with CFC from the erythroid line (56% and 72% of total CFC, respectively). Over 8 weeks, TPOFLK-stimulated liquid cultures gradually favored CFU-granulocyte macrophage (GM) expansion/maintenance, with CFC-erythroid exhausting from week 4 for Lin- cells and week 6 for CD133+ cells. However, from week 2 to week 8 of TPOFLK-supplemented liquid culture, Lin- HSPC yielded a higher ratio of CFC-GM than CD133+ cells (74% versus 58%, respectively, of total scored CFC) (Fig. 4).
Figure 4. Lin- cells preferentially expand CFC-GM in liquid culture stimulated by TPOFLK. After isolation, both Lin- and CD133+ cells were predominantly enriched with CFC-erythroid. However, when growing both cell subsets in TPOFLK-stimulated culture systems over 8 weeks, a higher proportion of CFC-GM was progressively expanded. Results are expressed as mean percentage of total CFC scored at each time point of four separate experiments.
Lin- Cells Contain More LTC-IC than CD133+ Cells and MNC
After 6 weeks in stroma-supported LTC assay, Lin- cells produced significantly more CFC than CD133+ cells (fivefold) and MNC (3,346-fold) (p < 0.05, n = 3) as shown by Figure 5. Taken together, these data showed that Lin- cells were enriched for more LTC-IC than CD133+ cells and MNC.
Figure 5. Lin- cells produced significantly more CFC in an LTC assay than MNC and CD133+ cells. After 6 weeks in an LTC system, Lin- cells scored a significantly higher cumulative CFC count than MNC and CD133+ cells (p < 0.05). When compared with respective CFC baseline seeded in the LTC assay, Lin- was the only cell population to expand CFC. Results are expressed as mean cumulative CFC count ± SE of three separate experiments.
DISCUSSION
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Tindle RW, Nichols RA, Chan L et al. A novel monoclonal antibody BI-3C5 recognises myeloblasts and non-B non-T lymphoblasts in acute leukaemias and CGL blast crises, and reacts with immature cells in normal bone marrow. Leuk Res 1985;9:1–9.
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Forraz N, Pettengell R, Deglesne PA et al. AC133 (+) umbilical cord blood progenitors demonstrate rapid self-renewal and low apoptosis. Br J Haematol 2002;119:516–524.
Osawa M, Hanada K, Hamada H et al. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996;273:242–245.
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b School of Life Sciences, Kingston University, Kingston upon Thames, UK;
c Department of Haematology, St. George’s Hospital Medical School, London, UK
Key Words. Negative selection ? Stem cells ? LTC-IC ? CD34 ? Ex vivo expansion
Colin P. McGuckin, Ph.D., Reader, School of Life Sciences, Faculty of Science, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, United Kingdom. Telephone: 44-797-126-6764; Fax: 44-208-725-0245; e-mail: c.mcguckin@kingston.ac.uk
ABSTRACT
Since the 1980s, hematopoietic stem cell transplantation and clinical research have mainly revolved around the cell surface protein, CD34, used as a marker for positive selection of heterogeneous hematopoietic stem and progenitor cells (HSPC) . Although the true role of the CD34 molecule continues to be debated, CD34+ HSPC have been functionally defined as capable of generating progenitor-derived clones in vitro and by their potential in reconstituting the lymphomyelopoietic system in myelocompromised hosts . CD133 was reported as an alternative marker for positive selection methods targeting more primitive HSPC enriched with CD34bright cells . However, we have recently reported that although CD133+ cells had interesting ex vivo expansion potential, they still encompassed cells at various stages of differentiation .
Several studies have demonstrated that cells that lack CD34 and hematopoietic lineage markers (Lin-/CD34-) could engraft immunocompromised animal hosts and sustain long-term in vivo hematopoiesis . Controversies then rapidly appeared by other groups suggesting limited hematopoietic engraftment potential to Lin-CD34- cells when compared with Lin-CD34+ HSPC . These investigators then hypothesized that small numbers of contaminating CD34+ HSPC could account for CD34- cell engraftment to the marrow . This controversy has been partly reconciled due to papers indicating the reversibility of CD34 expression, which may vary in vivo according to variable engraftment requirements .
Much of the problem with Lin- cell populations described so far is that they are poorly characterized. Modifications in previously described isolation protocols may also account for the variability of engraftment in immunocompromised animal models . Due to the need to find an optimized population, we have developed a reproducible strategy for primitive cell harvest.
MATERIALS AND METHODS
Cell Purification and Phenotypic Characterization
The mean purity of CD133+ immunomagnetically selected cells was 94.3% ± 0.9% and represented 0.4% ± 0.04% of the original UCB MNC (n = 19). CD133+ cells coexpressed other surface markers as follows: CD34 (93.8% ± 0.9%), CD38 (62.7% ± 3.7%), CD164 (63.8% ± 6.0%), CD162 (69.3% ± 2.6%), and CXCR4 (67.7% ± 1.4%).
Lin- HSPC were characterized as a discrete cell population representing 0.1% ± 0.02% (n = 11) of original UCB MNC. The Lin- cell subset was negative for a wide range of hematopoietic lineage markers, including CD45, glycophorin-A, CD38, CD7, CD33, CD56, CD16, CD3, and CD2. A proportion of Lin- HSPC nonetheless expressed markers: A) reflecting their immaturity status , such as CD133 (7.0% ± 0.8%), CD34 (14.4% ± 3.6%), intracellular CD34 (16.2% ± 0.6%), and CD164 (16.0% ± 4.1%), or B) that were involved in HSPC migration, adhesion, and homing to the bone marrow (BM) , CXCR4 (48.6% ± 4.0%), and CD162 (96.7% ± 2.0%) (Fig. 1). Our negative isolation protocol appeared to be highly reproducible and isolated a rare primitive Lin- cell population.
Figure 1. Phenotypic characterization of Lin- cells after isolation from UCB. Lin- cells expressed higher levels of immaturity markers, such as CD133, CD34 (n = 7), intracellular CD34 (n = 2), and CD164 (n = 5), than low-density UCB MNC. Lin- cells also expressed CXCR4 (n = 3) and CD162 (n = 3), both involved in hematopoietic progenitors homing to the BM. Results are expressed as percentage of the Lin- cell population ± SE.
CD133+ Cells Demonstrated a High Proliferation Potential in Cytokine-Stimulated Liquid Culture
Over 8 weeks in liquid culture, CD133+ cells proliferated more rapidly and yielded a significantly higher viable total cell number-fold increase (FI) than Lin- HSPC under both K36EG (p < 0.01) and TPOFLK (p < 0.05) stimulations (Fig. 2). In both culture systems, MNC were baseline and exhausted by week 7 of culture. Interestingly, TPOFLK cytokine mix induced a significantly higher viable cell FI when growing CD133+ cells for 8 weeks in culture (TPOFLK FI: 77.10 ± 0.27 x 107 versus K36EG FI: 1.53 ± 0.60 x 107, p < 0.05, n = 4). A similar pattern was observed for Lin- HSPC with a higher proliferation potential under TPOFLK synergism (week 8 FI: 33.20 ± 1.75) when compared with K36EG-stimulated liquid culture in which they stopped growing by week 6 (p < 0.001, n = 4). At first analysis, when compared with Lin- cells, CD133+ had a better ability to produce large cell numbers in TPOFLK-stimulated liquid culture.
Figure 2. CD133+ cells demonstrated a high proliferation potential in cytokine-stimulated liquid culture. Ex vivo expansion of Lin- cells, CD133+ cells, and MNC over 8 weeks in liquid culture stimulated either by TPOFLK or K36EG. Over 8 weeks, CD133+ cells proliferated more rapidly and yielded significantly higher cumulative CFC counts than Lin- cells under both K36EG (p < 0.01) and TPOFLK (p < 0.05) growth factor stimulations. When compared with K36EG stimulation, TPOFLK synergism induced significantly cumulative CFC counts for both CD133+ cells and Lin- cells. MNC did not expand CFC and exhausted by week 8. Results are expressed as mean ± SE of four separate experiments.
TPOFLK-Stimulated Liquid Culture of Lin- Cells Maintains a More Primitive Population of HSPC than CD133+ Cells
The TPOFLK-stimulated liquid culture expansion system consistently maintained a higher proportion of CD133+ and Lin- cell-derived primitive colony-forming cells (CFC) when compared with the effect K36EG cytokine mix on these cells (Fig. 3) (p < 0.05, n = 4). This confirmed the value of TPOFLK synergism when expanding primitive HSPC in liquid culture.
Figure 3. TPOFLK-stimulated liquid culture of Lin- cells maintains a more primitive population of HSPC than CD133+ cells. Lin-, CD133+, and MNC were respectively grown over 8 weeks in liquid culture stimulated either by TPOFLK or K36EG. At weeks 2, 4, 6, and 8, 104 cells from each cell type (Lin-, CD133+, and MNC), in both TPOFLK and K36EG conditions, were seeded in a clonogenic assay to enumerate CFC capacity. TPOFLK synergism consistently maintained a higher proportion of CD133+ and Lin- cell-derived CFC when compared with K36EG cytokine mix stimulation (p < 0.05). In TPOFLK-stimulated expansion system, Lin- cells produced more CFC/104 seeded cells than CD133+ cells (*p < 0.05). Results are expressed as mean ± SE of four separate experiments.
After immediate selection, CD133+ cells were enriched for more CFC/104-seeded cells (485 ± 111) when compared with Lin- cells (251 ± 41) CFC and MNC (12 ± 2) (p < 0.05, n = 5). However, in TPOFLK-stimulated liquid culture, Lin- cells consistently maintained a higher proportion of CFC at weeks 4, 6, and 8 than CD133+ cells and MNC (Fig. 3). This difference was particularly significant at 6 and 8 weeks (p < 0.05). Interestingly, in TPOFLK-stimulated liquid culture, the peak in CFC maintenance/expansion from Lin- cells was observed at 6 weeks of expansion (34 ± 8 CFC/104 cells), correlating with their slow proliferation pattern. Under the same conditions, the highest CFC frequency from CD133+ cells appeared earlier at week 4 and gradually faded by week 8 (Fig. 3).
After selection, both Lin- and CD133+ cells were predominantly enriched with CFC from the erythroid line (56% and 72% of total CFC, respectively). Over 8 weeks, TPOFLK-stimulated liquid cultures gradually favored CFU-granulocyte macrophage (GM) expansion/maintenance, with CFC-erythroid exhausting from week 4 for Lin- cells and week 6 for CD133+ cells. However, from week 2 to week 8 of TPOFLK-supplemented liquid culture, Lin- HSPC yielded a higher ratio of CFC-GM than CD133+ cells (74% versus 58%, respectively, of total scored CFC) (Fig. 4).
Figure 4. Lin- cells preferentially expand CFC-GM in liquid culture stimulated by TPOFLK. After isolation, both Lin- and CD133+ cells were predominantly enriched with CFC-erythroid. However, when growing both cell subsets in TPOFLK-stimulated culture systems over 8 weeks, a higher proportion of CFC-GM was progressively expanded. Results are expressed as mean percentage of total CFC scored at each time point of four separate experiments.
Lin- Cells Contain More LTC-IC than CD133+ Cells and MNC
After 6 weeks in stroma-supported LTC assay, Lin- cells produced significantly more CFC than CD133+ cells (fivefold) and MNC (3,346-fold) (p < 0.05, n = 3) as shown by Figure 5. Taken together, these data showed that Lin- cells were enriched for more LTC-IC than CD133+ cells and MNC.
Figure 5. Lin- cells produced significantly more CFC in an LTC assay than MNC and CD133+ cells. After 6 weeks in an LTC system, Lin- cells scored a significantly higher cumulative CFC count than MNC and CD133+ cells (p < 0.05). When compared with respective CFC baseline seeded in the LTC assay, Lin- was the only cell population to expand CFC. Results are expressed as mean cumulative CFC count ± SE of three separate experiments.
DISCUSSION
Civin CI, Strauss LC, Brovall C et al. Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J Immunol 1984;133:157–165.
Tindle RW, Nichols RA, Chan L et al. A novel monoclonal antibody BI-3C5 recognises myeloblasts and non-B non-T lymphoblasts in acute leukaemias and CGL blast crises, and reacts with immature cells in normal bone marrow. Leuk Res 1985;9:1–9.
Stella CC, Cazzola M, De Fabritiis P et al. CD34-positive cells: biology and clinical relevance. Haematologica 1995;80:367–387.
Kvalheim G. Clinical use of enriched CD34+ cells. Blood Ther Med 2002;2:57–64.
Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood 1993;81:2844–2853.
Pasino M, Lanza T, Marotta F et al. Flow cytometric and functional characterization of AC133(+) cells from human umbilical cord blood. Br J Haematol 2000;108:793–800.
Yin AH, Miraglia S, Zanjani ED et al. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 1997;90:5002–5012.
Forraz N, Pettengell R, Deglesne PA et al. AC133 (+) umbilical cord blood progenitors demonstrate rapid self-renewal and low apoptosis. Br J Haematol 2002;119:516–524.
Osawa M, Hanada K, Hamada H et al. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996;273:242–245.
Bhatia M, Bonnet D, Murdoch B et al. A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med 1998;4:1038–1045.
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