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Analysis of Primitive CD34– and CD34 Hematopoietic Cells from Adults: Gain and

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发表于 2009-3-5 10:37 |显示全部帖子
a Department of Molecular Microbiology and Immunology, Oregon Health and Sciences University, Portland, Oregon, USA;% z% d: X4 W* H1 x9 Y
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b Stem Cell Research Laboratory, Pacific Northwest Regional Blood Services, American Red Cross, Portland, Oregon, USA;
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- S$ T% ~" {, Cc Department of Pathology, University of Virginia, Charlottesville, Virginia, USA' D& K4 L  R6 Y' E
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Key Words. CD34  cells ? CD34–cells ? Cell surface markers ? Cell culture ? Expansion Flow cytometry ? Hematopoiesis ? Hemopoietic cells
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Correspondence: Douglas C. Dooley, Ph.D., Department of Molecular Microbiology and Immunology, Oregon Health and Sciences University, 3181 SW Sam Jackson Park Road, L220, Portland, Oregon 97201-3098, USA. Telephone: 503-494-5312; Fax: 503-494-6862; e-mail: dooleyd@ohsu.edu/ o3 @* Z; k: n/ o6 P# j
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ABSTRACT
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CD34 antigen, a transmembrane glycoprotein, is a member of the sialomucin family . The antigen was initially discovered on a myeloblastic leukemia cell line . While the precise function of CD34 is unknown, involvement in either cytoadhesion or differentiation (or both) has been suggested . Lacking its own kinase activity, transduction of signals from CD34 may involve the intracellular adapter protein CrkL . Due to its expression in early hematopoietic development, the antigen has become a valuable tool for identification and purification of human hematopoietic cells . Thus, it was surprising to learn in 1996 that CD34 expression in mice was not mandatory for survival  and that murine hematopoiesis could be reconstituted by CD34– LIN– cells . CD34– hematopoietic cells were subsequently identified in other species . Engraftment activities were demonstrated in CD34– fractions of human bone marrow, mobilized peripheral blood progenitor cell (PBPC) products, and umbilical cord blood (UCB) .: {# }5 j3 J0 m; [7 z+ D

% V' m, j, u/ l- x. }4 ZIn vitro culture systems have been used to investigate human CD34– cells and their modulation of CD34 antigen. Those experiments proved difficult because of the limited growth potential of CD34– UCB and marrow cells in stroma-free culture . Nonetheless, data suggested that colony-forming activity and CD34 antigen expression were present after 7–10 days’cultivation . Experiments in murine systems showed that CD34– stem cells expressed CD34 after mobilization or treatment with 5-fluorouracil . Taken together, those results suggested an association between cellular activation (cytokine responsiveness) and upregulation of CD34. However, the exact nature of "cellular activation" has been questioned , and a direct link between proliferation and CD34 expression has not been shown. Other questions remain. The kinetics of upregulation are not well defined, and it is uncertain whether CD34– cells can divide prior to upregulating CD34 . Furthermore, it is not clear if all cells or only growing cells in a CD34– culture upregulate CD34. Last, relatively little information is available about the properties of CD34– cells from PBPC products . Since PBPC represents an important source of stem cells for transplantation, characterization of circulating adult CD34– cells is important., f* F* }' K4 Y! \! U9 x" b$ }
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For this report, we chose to study the CD34– CD38– LIN– (CD34– ) subset, since this is a particularly primitive hematopoietic fraction . Mobilized CD34– and CD34 CD38– LIN– (CD34 ) cells were purified, and their proliferative potentials were characterized. CD34 antigen modulation was examined, and the relationship of CD34 expression to cell proliferation and growth potential were directly determined. Here we report that both CD34– and CD34  cells modulate CD34 antigen expression shortly after initiation of culture. In both cases, modulation of CD34 reflects the proliferative status of LIN– cells in culture.
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MATERIALS AND METHODS+ p# t: X) d: ]  y' c7 P
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Isolation of CD34– Hematopoietic Cells' x# G$ ~0 R2 s* D+ {- c/ @& |% X

0 l9 l! r( T7 W% {$ G" W2 L4 k4 oThe frequencies of CD34– CD38– LIN– (CD34– ) and CD34 CD38– LIN– (CD34 ) cells in PBPC were too low to be measured accurately. However, they were readily detected in the LIN-depleted fraction where CD34– cells were significantly more numerous than CD34  cells (4.6% versus 1.5%, p
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3 [* i4 H9 o6 F$ H* a5 d% T, D, p# zTable 1. Frequency (%) of primitive hematopoietic cells in PBPC after depletion of LIN  cells" e& [( I* l/ H$ }4 z
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Figure 1 illustrates the purification of CD34– and CD34  fractions by flow cytometric sorting. To achieve high purity, narrow gates were used to capture cells with very low expression of CD34 and LIN. Cells of interest were low in side scatter, low to moderate in forward scatter, LIN– , in the lowest 10% in CD38 fluorescence, and either CD34BRIGHT or CD34– (gates D and F, Fig. 1, Panel II). Cells lacking CD34 expression were defined as those with less than the modal brightness of the PE-Cy5 isotypic control. The higher frequency of CD34– than CD34  cells is apparent in Figure 1. Reanalysis of the CD34– fraction showed that more than 97% of the cells were CD34– . CD34– cells from PBPC were noticeably smaller than corresponding CD34  cells . Data presented below (Figures 3, 5, 6, Tables 2, 3) and RT-PCR analyses (not shown) confirmed that CD34  and CD34– fractions were well separated.. h5 B: d5 D. ?& w; [/ Z
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Figure 1. Flow cytometric sorting of CD34– and CD34  fractions from lineage-depleted PBPC. Flow cytometric profiles obtained from LIN-depleted adult cells. Panel I: Isotypic control. Panel II: Gating strategy for purification of CD34– and CD34  fractions. LIN– events (Region C) were analyzed for expression of CD34 and CD38 (lower left). The F and D gates captured CD34– cells and CD34  cells, respectively. In this display, events with very low CD34 and CD38 fluorescence rest on the axes and are not visible. The Baseline Offset function was applied to the same data to enhance the display of low fluorescence events (lower right). This function randomizes-low level signals in a Gaussian distribution across the first decade. Baseline Offset was also used to improve visualization of the single-parameter CD38 histogram in Panel I.% i1 O& y' \% y; J7 M" w

; H) M+ A' ~9 a: A6 }6 X$ A% `! IFigure 3. Expression of CD34 antigen by LIN– cells in PKHBRIGHT and PKHINTERMED fractions. CD34– and CD34  cells were stained with PKH26, cultured for 48 hours, relabeled with antibodies, and analyzed. Lineage commitment profiles were almost identical: 63% LIN– in the CD34– culture (upper left) and 62% in the CD34  culture. LIN– populations were analyzed for PKH26 fluorescence (upper right). Three PKH profiles are displayed: (1) Dashed line: Day zero cells. (2) Shaded peak: Day 2, CD34– culture. (3) Dotted line: Day 2CD34  culture. Region R1 defines Day 0 PKHBRIGHT fluorescence, while R2 includes growing PKHINTERMED cells.
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Figure 5. Proliferative status and CD34 expression in early cells. CD34– and CD34  PBPCs were cultured for 42 hours and analyzed for proliferative state. (A): Freshly isolated CD34  and CD34– fractions were reanalyzed for expression of CD34 (left) and Ki67 (right). Plots show isotype controls (front rows), the CD34– isolate (middle rows), and the CD34  isolate (back rows). (B): After cultivation, cells from the CD34– culture were analyzed for CD34 and LIN phenotypes. The cellular distribution into the four regions was as follows: R1 = 17%; R2 = 74%; R3 = 3.1%; R4 = 0.6%. Ki67 and PY profiles of each region are shown on the right. (C): Postcultivation phenotype of the CD34  culture, where R1 = 1.3%; R2 = 14%; R3 = 82%; R4 = 0.7%. Proliferative activities shown on the right. CD34 and LIN phenotypes obtained after Ki67 staining (permeablization) or PY staining were very similar. A second experiment gave similar results. Abbreviations: PBPC, peripheral blood progenitor cell; PY, pyronin Y.9 t1 [0 @; [6 J2 {+ e
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Figure 6. Isolation of primitive cells that did or did not modulate CD34 expression. PKH26-stained, CD34– , and CD34  cultures were collected after 3 days, relabeled with antibodies, and subjected to sorting. The PKHBRIGHT gate was placed at the position of day zero PKH fluorescence. Panel I: In the CD34– culture, the PKHBRIGHT LIN– CD38– population was separated into fractions that were still CD34– (Region B1) or had upregulated CD34 (Region B2). Panel II: Analogously, in the CD34  culture, the PKHBRIGHT LIN– CD38– fraction was separated into cells that had retained CD34 expression (Region B4) or those that had lost CD34 (Region B3). The four fractions were returned to culture (Fig. 7). Results are typical of four independent trials." {% h" [( k% }6 }5 A1 o

! I6 T/ Y1 H2 u8 V+ g4 e- K8 jTable 2. Phenotypic analysis of PKHBRIGHT and PKHINTERMED fractions (% of cells in each column with indicated phenotype) in CD34– and CD34  cultures after short-term incubation1 F7 p: |# E' t  t  D
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Table 3. CD34– cells initiate growth more slowly than do CD34  cells9 r, g7 I. J: Y( ]+ d

  n, u' [$ c! F9 uIn Vitro Proliferative Activity of CD34– Cells' r( Y0 D( Q% i; P

# p0 Q& S: f6 H8 X2 {) SColony-forming cell (CFC) activity of CD34– and CD34  fractions was assessed in serum-free methylcellulose containing six cytokines. No significant differences in CFC frequencies could be detected between the CD34– fraction and the CD34  fraction (7%   7% and 10%   11%, respectively; n = 11, paired t-test, p = .29). The distribution of progenitor lineages in the CD34– fraction (55% colony-forming units-granulocyte-macrophage (CFU-GM), 37% BFUE, and 9% colony-forming units-mixed (CFU-Mix) was similar to that observed in the CD34  population (49%, 36%, and 13%, respectively).# k0 v. O: V# Z1 F  K
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Suspension cultures of PBPC CD34– and CD34  fractions proved very active, as both expanded approximately 10,000-fold (Fig. 2A). Over 8 weeks, the fractions generated similar numbers of CD34  cells (Fig. 2D). CD34  cells appeared in CD34– cultures within just 2 days. After 6 days of cultivation, CD34  cell frequencies in CD34– and CD34 cul-tures were similar (Fig. 2E). Each CD34– cell produced 630 ± 770 CD34  cells, while each CD34  cell produced 1,600 ± 1,800 CD34  cells, a difference that did not reach significance (n = 5, p = .13, paired t-test).
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; x! N- ~5 |; d) N! {4 nFigure 2. Ex vivo proliferation of CD34– and CD34  fractions from PBPC. Purified CD34– cells and CD34  cells were cultured in serum-free medium supplemented with IL-3, IL-6, SCF, FL, and TPO. (A): Average cell number per suspension culture (n = 6). Little difference in cell production was observed between CD34– and CD34  fractions. (B): Average number of progenitors produced per CD34– and CD34  suspension culture (n = 7). (C): CD34– and CD34  fractions were cultured over murine S-17 stromal layers (n = 3). Assessment of nonadherent fractions suggests the presence of extended LTCIC in both populations. (D): Average absolute number of CD34  cells per suspension culture. Generation of CD34  cells continued for at least 7 weeks, with little difference between CD34  and CD34– cultures. (E): Absolute frequencies of CD34  cells in the same CD34– and CD34  cultures. Data points offset by one day for clar-; K2 D, ]2 p' {4 G
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The prolonged generation of CD34  cells in CD34– suspension cultures was confirmed in progenitor assays. In CD34– cultures, output of both CFU-GM and CD34  cells peaked after 7–8 weeks (Fig. 2B, 2D). Similar numbers of progenitors were generated per initial cell (51 ± 63 CFC in CD34– suspensions versus 47 ± 59 CFC in CD34  suspensions). In stroma-based cultures, both populations generated CFC for more than 80 days (Fig. 2C), demonstrating the presence of long-term culture-initiating cells (LTCIC) and extended LTCIC. Collectively, these data demonstrated that CD34– and CD34  fractions from adult PBPC collections exhibited similar proliferative capabilities when cultured in vitro.
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$ l/ ~6 z: z6 P: O. q$ `) m& mModulation of CD34 Antigen Expression, x* Z% @& c& E8 G6 Q

/ z9 L; l7 ^5 W$ dThe ready growth of CD34– PBPCs provided an opportunity to examine the relationship between cell proliferation and expression of CD34 antigen. In these studies, attention was focused on the response of the most primitive (LIN– ) cells in the culture. PKH26- or PKH67-loaded CD34– and CD34  cells were cultured for 48–72 hours, harvested, and restained with antibodies. Cells were analyzed for proliferation and antigen expression. Cells that had undergone little or no growth were PKHBRIGHT, while those that had completed several division cycles were PKHINTERMED.; b) T, h5 y. K
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Figure 3 (top) shows typical PKH profiles of LIN– cells in CD34– and CD34  cultures. Gate R1 delineates PKHBRIGHT (day zero) fluorescence, while R2 marks the PKHINTERMED region. R2 was placed so that cells with modal brightness in R1 would have to divide twice to reach R2. In the CD34– culture (central panels), only 6% of non-proliferating LIN– PKHBRIGHT cells had upregulated CD34, while 10 times as many (59%) expressed CD34 in the proliferating LIN– PKHINTERMED fraction. For comparison, the bottom panels of Figure 3 present the companion CD34  culture. In the proliferating LIN– PKHINTERMED fraction, 89% continued to express CD34. Surprisingly, 37% of the undifferentiated LIN– PKHBRIGHT cells in the CD34  culture lost CD34 expression.2 Y  T4 ~2 B3 [$ ~

2 U5 ~+ s3 L& X: Y# B8 iThese data suggest a correlation between CD34 expression and cell proliferation. That inference is strengthened by Table 2, which summarizes data from seven similar trials. For example, in CD34– cultures, 32% of growing cells expressed CD34 (column 2), compared with only 7% of PKHBRIGHT cells (column 1), and these values were significantly different (p ( b4 A- k7 K  W; ]

/ X  |' n3 P5 E+ F, H- V0 {6 J1 A% WFrom the preceding data, it was not possible to state unequivocally that PKHBRIGHT cells were quiescent. To resolve that question, cells were stained with pyronin Y (PY). Quiescent cells are PYLOW, whereas cycling cells are PYHIGH . This was confirmed with fresh (inherently quiescent) CD34  CD38– PBPCs, 75%   11% of which were PYLOW (n = 5). In the following experiments, PKH-stained, CD34 CD38– PBPC cells were cultured for 4–6 days. Upon flow analysis, four populations were identified: PKHBRIGHTCD34  (Fraction A), PKHBRIGHTCD34– (Fraction B), PKHINTERMEDCD34  (Fraction C), and PKHINTERMEDCD34– (Fraction D). The analysis of those populations for PY content and CD38 expression is presented in Figure 4. In the PKHBRIGHT fractions (Fig. 4A, B), 80%–90% of cells were PYLOW. In contrast, 70%–80% of PKHINTERMED cells were PYHIGH (Fig. 4C, D). This confirmed that PKHBRIGHT cells were quiescent, while PKHINTERMED cells were actively cycling. It is notable that PKHBRIGHT cells that expressed CD34 were predominately CD38 , while PKHBRIGHTCD34– cells were primarily CD38– (see Discussion).
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( G1 M: S& n& \( Z* W- ?- Z6 i, LFigure 4. Pyronin Y (PY) analysis of PKHBRIGHT and PKHINTERMED cells from CD34  suspension cultures. CD34  CD38– cells from PBPC were stained with PKH67 and cultured 4–6 days (n = 6). Four cell populations were analyzed for PY content and expression of CD38 antigen: (A) PKHBRIGHT CD34 , (B) PKHBRIGHT CD34– , (C) PKHINTERMED CD34 , and (D) PKHINTERMED CD34– . A through D constituted an average of 7%, 5%, 22%, and 65% (respectively) of total cells. Figure displays the distribution of pyronin and CD38 phenotypes in each of the four populations (p
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The proliferative status of cultured cells was also determined by assessing their expression of Ki67 antigen. Ki67 is absent from G0 cells but present in proliferating cells in all phases of the cell cycle . Preliminary studies showed that staining with Ki67 and PY yielded comparable results (data not shown). Starting with freshly sorted CD34– and CD34  fractions, cells were examined for expression of Ki67 and CD34. Analysis confirmed clean separation of CD34– and CD34  populations. Both populations were Ki67LOW (Fig. 5A). CD34– and CD34  fractions were cultured for 42 hours and analyzed for antigen expression and proliferative status. In the CD34– culture (Fig. 5B), most LIN– cells were CD34LOW (R2) or CD34– (R1). In the CD34  culture (Fig. 5C), the bulk of LIN– cells were still CD34BRIGHT (R3); however, early dimming of CD34 in LIN– cells was apparent. In both CD34– and CD34  cultures, Ki67 and PY analysis shows that CD34– cells (R1) were quiescent, while CD34BRIGHT cells (R3) were proliferating. Interestingly, in the CD34LOW fractions (R2), low levels of PY staining and Ki67 expression were seen. These data confirmed the correlation between CD34 antigen expression and proliferation in both CD34  and CD34– cultures.9 X/ q' ?" ^& f: Q4 z4 m

: E! s# ^0 U1 ?3 e9 S, M8 n2 wInitial CD34 Phenotype and the Onset of Cell Proliferation
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$ x* L- `0 x- Z( k6 gFigures 3, 5, and 6 suggest that, on average, CD34– cells initiated growth more slowly than did CD34  cells. To test the generality of that observation, PKH26 profiles from CD34– and CD34  cultures were analyzed for the proportion of cells that were PKHBRIGHT and those that had undergone greater than three division cycles. After 2 days of cultivation, the proportion of cells that had undergone at least three divisions was twice as great in CD34  cultures as in CD34– cultures (p
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Modulation of CD34 Expression: Relation to Growth Potential
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+ E9 @* n5 h; K& K; y5 D) ?Finally, we asked if cells that rapidly modulate CD34 antigen in culture might differ from those that retain their original phenotype. As one approach, we studied the possibility that modulating and nonmodulating cells might differ in their capacity for long-term proliferation. Since it was important to minimize confounding parameters (differentiation and growth-related loss of proliferative potential), analysis was limited to LIN– PKHBRIGHT cells. In a typical trial, CD34– and CD34  cells were cultured for 3 days, then harvested and sorted for LIN– CD38– PKHBRIGHT subpopulations differing in CD34 expression. In the CD34– culture, two populations were resolved (Fig. 6, Panel I): B1, which remained CD34– , and B2, which had upregulated CD34 expression. Two fractions were isolated from the CD34  culture (Panel II): B3, which had downregulated CD34 expression, and B4, which remained CD34 . From preceding considerations (Figs. 3, 5), it was anticipated that fraction B2 cells would be rare. Indeed, B2 represented only 1% of PKHBRIGHT LIN– cells. Nevertheless, sufficient cells were obtained for analysis.1 ]3 k, f' p" d) [
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Fractions B1–B4, quiescent and LIN– , were resuspended in serum-free culture to assess in vitro proliferative potential. Average expansion of the fractions ranged from 350,000-fold to 730,000-fold, with no significant differences among them (n = 4). The kinetics and total number of CD34  cells and CFC generated were similar (Fig. 7). Thus, neither rapid up-modulation nor down-modulation of CD34 antigen distinguished subpopulations that differed in growth capacity measured in vitro./ g9 t/ \9 x" k" S7 i! y" V9 f
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Figure 7. Proliferative activity of primitive cells that did or did not modulate CD34 expression. Fractions B1–B4, defined in Figure 6, were assessed for their abilities to generate CD34  cells (top) and CFC (bottom). Data pooled from four separate trials. All four fractions were hematopoietically active in suspension culture for >8 weeks. No consistent differences were observed. Abbreviation: CFU, colony-forming unit.
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DISCUSSION
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This research was supported by the American Red Cross.3 |  V, l0 Q  l1 q  |, z

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Kato S, Ando K, Nakamura Y et al. Absence of a CD34-hematopoietic precursor population in recipients of CD34  stem cell transplantation. Bone Marrow Transpl 2001;28:587–595.+ ^1 u  Q& S; O8 U( c

! U* P) Z8 F( v- C0 aTajima F, Deguchi T, Laver J et al. Reciprocal expression of CD38 and CD34 by adult murine hematopoietic stem cells. Blood 2001;97:2618–2624.
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干细胞研究人员的天堂

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我该不会是最后一个顶的吧  

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干细胞之家微信公众号
端粒酶研究

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这个贴好像之前没见过  

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活着,以死的姿态……  

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来几句吧  

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爷爷都是从孙子走过来的。  

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加油啊!!!!顶哦!!!!!  

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既然来了,就留个脚印  
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