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a King-George Laboratory, St. George’s Hospital Medical School and Kingston University, London, UK;# s. W: @& m" ^* g3 m: Z( K( D
7 C! M+ b3 C: L v. {5 x- N# nb School of Life Sciences, Kingston University, Kingston upon Thames, UK;7 z0 }+ I7 p4 M, C( W
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c Department of Haematology, St. George’s Hospital Medical School, London, UK, `9 ~9 ?4 s/ W+ {
, N3 ?5 G% Z1 i. JKey Words. Negative selection ? Stem cells ? LTC-IC ? CD34 ? Ex vivo expansion
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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$ |. R/ U* I* L+ T9 L& b* S
; S2 S# }% R" ~5 K- jABSTRACT
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: | L' g* b+ C K1 Z9 uSince 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 .
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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 .) ]! P$ V* d6 M1 s4 S/ i+ X% _
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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.* J' `! F" d/ H4 T( [' J- A
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MATERIALS AND METHODS
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Cell Purification and Phenotypic Characterization
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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%).: f2 ~- j6 e+ R* e9 N6 ^
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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.
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; X2 k- Z) G" s7 hFigure 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.
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3 v3 ?" y) R* wCD133 Cells Demonstrated a High Proliferation Potential in Cytokine-Stimulated Liquid Culture
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3 l6 `7 ]! o/ Y( I6 \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
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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 ( e! @( O' T8 v! Z
9 q! }' U9 D- Z) @TPOFLK-Stimulated Liquid Culture of Lin- Cells Maintains a More Primitive Population of HSPC than CD133 Cells6 L$ Q% `$ t2 i4 O8 M9 \3 `+ z
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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
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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 8 z/ A+ w) o% a# [
+ q* D3 a o0 o; MAfter 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 1 y* m5 y& _0 } E; l
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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).
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' B: y }* v; EFigure 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.
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Lin- Cells Contain More LTC-IC than CD133 Cells and MNC
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After 6 weeks in stroma-supported LTC assay, Lin- cells produced significantly more CFC than CD133 cells (fivefold) and MNC (3,346-fold) (p 2 n% Y5 s) P9 ]0 {6 A
5 r2 i+ h% [+ C, Z9 RFigure 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
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