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Stem Cells 2004;22:556-569 www.StemCells.com
© 2004 AlphaMed Press

Analysis of Primitive CD34– and CD34+ Hematopoietic Cells from Adults: Gain and Loss of CD34 Antigen by Undifferentiated Cells Are Closely Linked to Proliferative Status in Culture

Douglas C. Dooleya, Barbara K. Oppenlanderb, Mang Xiaoc

a Department of Molecular Microbiology and Immunology, Oregon Health and Sciences University, Portland, Oregon, USA;
b Stem Cell Research Laboratory, Pacific Northwest Regional Blood Services, American Red Cross, Portland, Oregon, USA;
c Department of Pathology, University of Virginia, Charlottesville, Virginia, USA

Key Words. CD34+ cells • CD34–cells • Cell surface markers • Cell culture • Expansion Flow cytometry • Hematopoiesis • Hemopoietic cells

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{at}ohsu.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is limited understanding of CD34– hematopoietic cells and the linkage between CD34 antigen expression and cell proliferation. In this study, early CD34– CD38– LIN– (CD34– ) cells were purified from mobilized adult peripheral blood and carefully analyzed in vitro for growth and modulation of CD34. Mobilized CD34+CD38– LIN– (CD34+) cells were used for comparison. Expression of CD34, CD38, and LIN antigens was determined, and proliferative responses were assessed with PKH tracking dye, expression of Ki67 antigen, and uptake of pyronin Y. Suspension cultures of adult CD34– cells generated CD34+ cells and progenitors for >8 weeks. Stromal cultures demonstrated the presence of long-term culture-initiating cells within the CD34– fraction. While CD34– cells were slower to initiate growth than the CD34+ cells were, no significant difference in hematopoietic cell output was found. Upon cultivation of CD34– cells, CD34 antigen appeared within 48 hours but was restricted to those cells that had initiated growth. Surprisingly, CD34+ precursors lost CD34 expression in culture if they remained in G0 for more than 2 days. Those cells later regained expression of CD34 antigen upon initiation of growth. Comparison of cells that did or did not rapidly modulate CD34 antigen revealed no differences in long-term growth potential. In conclusion, in vitro expression of CD34 by CD34– and CD34+ populations is tightly linked to cellular proliferation. In this culture system, expression of CD34 antigen by LIN– cells constitutes an early hallmark of growth. Measurement of CD34 expression by LIN– cells in expansion culture underestimates the total content of hematopoietic cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD34 antigen, a transmembrane glycoprotein, is a member of the sialomucin family [1]. The antigen was initially discovered on a myeloblastic leukemia cell line [2]. While the precise function of CD34 is unknown, involvement in either cytoadhesion or differentiation (or both) has been suggested [1,3]. Lacking its own kinase activity, transduction of signals from CD34 may involve the intracellular adapter protein CrkL [4]. Due to its expression in early hematopoietic development, the antigen has become a valuable tool for identification and purification of human hematopoietic cells [5–8]. Thus, it was surprising to learn in 1996 that CD34 expression in mice was not mandatory for survival [9] and that murine hematopoiesis could be reconstituted by CD34– LIN– cells [10–14]. CD34– hematopoietic cells were subsequently identified in other species [15–17]. Engraftment activities were demonstrated in CD34– fractions of human bone marrow, mobilized peripheral blood progenitor cell (PBPC) products, and umbilical cord blood (UCB) [18–21; reviewed in 22–24].

In 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 [21, 23, 25–29]. Nonetheless, data suggested that colony-forming activity and CD34 antigen expression were present after 7–10 days’cultivation [17, 26, 29, 30]. Experiments in murine systems showed that CD34– stem cells expressed CD34 after mobilization or treatment with 5-fluorouracil [11, 31–33]. 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 [34], 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 [35]. 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 [17]. Since PBPC represents an important source of stem cells for transplantation, characterization of circulating adult CD34– cells is important.

For this report, we chose to study the CD34– CD38– LIN– (CD34– ) subset, since this is a particularly primitive hematopoietic fraction [21]. 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.


   MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
PBPCs were collected following treatment with granulocyte colony-stimulating factor with or without chemotherapy. Samples were held in liquid nitrogen until selected for analysis. Samples were obtained with approval of the American Red Cross Committee for the Protection of Human Subjects.

Flow Cytometric Analysis and Isolation of Primitive Hematopoietic Progenitors
This laboratory’s method for lineage depletion and flow cytometric sorting has been described in detail [36–39]. In brief, thawed PBPCs were enriched for LIN– cells using immunomagnetic separation (Stem Cell Technologies Inc., Vancouver, Canada) targeted against glycophorin A, CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b plus added CD41. The LIN– cell-enriched fraction was labeled with CD34 PE-Cy5 (clone 581, class III, Coulter-Immunotech Co., Miami, FL), fluorescein-6-isothiocyanate (FITC)–conjugated antibodies against LIN antigens (CD3, CD14, CD16, CD19, CD20, and CD56; Becton Dickinson Immunocytometry Systems, San Jose, CA), as well as CD42a-FITC and CD38 APC (Becton Dickinson) or isotypic control antibodies (from the corresponding vendor). Cells were sorted using a Coulter ELITE ESP unit, using propidium iodide (PI) as a viability marker (if PKH26 were not present). For data analysis, at least 10,000 events were collected. Reanalysis of sorted cells indicated >97% purity.

Sorting and Analysis of Cells Stained with Tracking Dyes
Suspensions enriched for LIN– cells were stained with PKH26 or PKH67 (Sigma Immunochemicals, St. Louis, MO) [39]. PKHBRIGHT cells were sorted into CD34– and CD34+ fractions. An aliquot of unsorted PKH-stained cells was treated with 1.0% paraformaldehyde and then stored at 4°C for subsequent reference of initial PKH fluorescence. Sorted cells were placed in suspension culture for the indicated period of time, and samples were collected for flow cytometric determination of PKH fluorescence and immunophenotype after restaining. In some cases, cultured cells were resorted and returned to culture. Where indicated, cellular RNA was stained with pyronin Y, as described by Ladd et al. [40] including the preincubation step with Hoechst 33342, which blocks pyronin Y staining of DNA. This stain distinguishes quiescent G0 cells (PYLOW) from actively cycling cells (PYHIGH) [40,41]. Alternatively, cells were permeabilized and stained with Ki67 antibody following manufacturer’s instructions (Becton Dickinson).

Suspension and Stromal Cultures
For stroma-free, serum-free suspension cultures, sorted cells were incubated in 24 well trays or T-25 flasks (Corning Inc., Corning, NY) containing 0.5 to 1.0 mL/well or 10 mL/flask serum-free expansion medium (SFEM; Stem Cell Technologies, Inc.) containing penicillin or streptomycin, low-density lipoproteins (Sigma-Aldrich, St. Louis, MO), recombinant human (rh) stem cell factor (SCF; rhSCF 50 ng/mL), Flt3 ligand (rhFL; 50 ng/mL), interleukin-3 (rhIL-3; 12.5 ng/mL), interleukin-6 (rhIL-6; 10 ng/mL), and thrombopoietin (rhTPO; 100 ng/mL). SCF was a generous gift from Amgen (Thousand Oaks, CA); FL, IL-3, and IL-6 were generously provided by Immunex Corporation (Seattle, WA). TPO was purchased from Genzyme Corporation (Cambridge, MA). CD34– cultures were initiated with an average of 82,000 cells (range: 1,800–183,000, depending on sort yield) and CD34+ cultures, with an average of 53,000 cells (range: 900–154,000). In stromal cultures, the murine cell line S-17 (known to support myeloid and lymphoid differentiation) was used. In this case, the medium consisted of Iscove’s modified Dulbecco’s medium (IMDM), 30% fetal bovine serum (FBS), 10% bovine serum albumin (BSA), 10– 4 M mercaptoethanol, 1% penicillin or streptomycin, 2 mM L-glutamine, 10– 6 M hydrocortisone hemisuccinate, and the same cytokines used in suspension culture. Cytokines were replenished once per week during demi-depopulation of stromal cultures and two to three times per week in liquid cultures. In rapidly expanding suspension cultures, cells were removed weekly to return the cell concentration to ~105 cells/mL. Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.

Colony Formation Assay
Committed hematopoietic progenitors from stromal cultures were quantitated in standard semisolid cultures in triplicate, using 1 mL of Methocult GF+ (#H4435, Stem Cell Technologies, Inc.), which consists of 1% methylcellulose in IMDM, 30% FBS, 1% BSA, 2 mM L-glutamine, 10– 4 M 2-mercaptoethanol, 50 ng/mL SCF, 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 20 ng/mL granulocyte colony-stimulating factor (G-CSF), 20 ng/mL IL-3, 20 ng/mL rhIL-6, and 3 U/mL erythropoietin. Nonadherent cells from stromal cultures were washed twice with IMDM/2% FBS before plating. Progenitors from serum-free suspension cultures were quantitated in triplicate using 1 mL of serum-free Methocult SFbit (#H4436, Stem Cell Technologies, Inc.) containing 1% methylcellulose in IMDM, 1% BSA, 2 mM L-glutamine, 10– 4 M 2-mercap-toethanol, 10 µg/mL bovine pancreatic insulin, 200 µg/mL human transferriniron saturated plus the same six cytokines contained in methylcell H4435. Cells from liquid culture were washed twice with SFEM before assay. Plates were scored for erythroid, granulocyte-macrophage, and mixed-lineage colonies after culturing for 14 days at 37°C, 5% CO2.

RT/PCR Analysis of CD34 Gene Expression
Cells were lysed in dimethyl pyrocarbonate–treated water containing 0.8% NP-40, 5 mM dithiothreitol (DTT), 1 unit ribonuclease inhibitor (RNasin), and 1 µg transfer RNA (Sigma). Supernatants from the lysates were digested with 1 U Deoxyribonuclease. Seven-microliter aliquots were reverse transcribed with GeneAmp RNA PCR kits (Perkin Elmer, Rockford, IL) and random hexamers (2.5 µM) as primers, RNase inhibitor (20 units) and murine leukemia virus reverse transcriptase (50 units). Amplification was conducted with the PCR core kit (Perkin Elmer) using 35 cycles of 1 minute denaturation at 94°C, 1 minute annealing at 65°C, and 2 minutes synthesis at 72°C. Oligonucleotides for CD34 gene expression analysis were sense primer 5'-AGGTATGCTCCCTGCTCCTGGCCC-3' and antisense primer 5'-AAGAACAGCCTCTGAGGTGTGTGC-3'. Sense primer 5'-GGTCGGAGTCAACGGATTTGG-3' and antisense primer 5'-TGTGGGCCAT GAGGTCCACCAC-3' were used as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reference. As a negative control, reverse transcriptase was omitted. Twenty microliters of the amplified products were run in 2% agarose gels and stained with ethidiumbromide dye.

Analysis of Data
In stroma-free and stroma-based cultures, the starting cell number was normalized to 10,000 cells to allow pooling of data between experiments. In stroma-free cultures (but not in stroma-based cultures) the weekly total viable cell count was corrected for periodic removal of cells at the time of medium change. To determine the proportion of cells having undergone 0, 1. . . . n cell divisions, PKH26 profiles were analyzed with ModFit LT software (Verity Software House, Topsham, ME). Significance of differences was determined with paired t-tests or Wilcoxon Signed Rank test, as indicated (Sigma Stat [SMSS Inc., Chicago, IL] or GB Stat [Dynamic Microsystems, Inc., Silver Spring, MD]).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of CD34– Hematopoietic Cells
The 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 < .009) (Table 1Go). These data compare favorably with values of 6% versus 3% published recently [17].


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  		Table 1. Frequency (%) of primitive hematopoietic cells in PBPC after depletion of LIN+ cells
 
Figure 1Go 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. 1Go, 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 1Go. 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 [29]. Data presented below (Figures 3Go, 5Go, 6Go, Tables 2Go, 3Go) and RT-PCR analyses (not shown) confirmed that CD34+ and CD34– fractions were well separated.



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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.

 


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  		Figure 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.

 


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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. 7Go). Results are typical of four independent trials.

 

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  		Table 2. Phenotypic analysis of PKHBRIGHT and PKHINTERMED fractions (% of cells in each column with indicated phenotype) in CD34– and CD34+ cultures after short-term incubation
 

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  		Table 3. CD34– cells initiate growth more slowly than do CD34+ cells
 
In Vitro Proliferative Activity of CD34– Cells
Colony-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).

Suspension cultures of PBPC CD34– and CD34+ fractions proved very active, as both expanded approximately 10,000-fold (Fig. 2AGo). Over 8 weeks, the fractions generated similar numbers of CD34+ cells (Fig. 2DGo). 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. 2EGo). 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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  		Figure 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-

 
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, 2DGo). 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. 2CGo), 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.

Modulation of CD34 Antigen Expression
The 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.

Figure 3Go (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 3Go 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.

These data suggest a correlation between CD34 expression and cell proliferation. That inference is strengthened by Table 2Go, 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 <.02). Conversely, both the CD34– LIN– fraction and the CD34– CD38– LIN– fraction were enriched in the PKHBRIGHT population relative to the PKHINTERMED population (column 1 versus 2, both fractions p < .02). A similar association between CD34 expression and proliferation was observed in CD34+ cultures. CD34 expression was higher in PKHINTERMED cells than in PKHBRIGHT cells (column 4 versus 3), while CD34– LIN– cells and CD34– cells were more highly enriched in the PKHBRIGHT fraction than in the PKHINTERMED fraction (column 3 versus 4). There was no difference in the proportions of growing cells in CD34– and CD34+ cultures that expressed CD34 (32% versus 47%). In summary, primitive CD34– cells were enriched in the PKHBRIGHT fractions of both CD34– and CD34+cultures; in contrast, CD34+ cells were enriched in the PKHINTERMED fractions.

From 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 [40,41]. 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 4Go. In the PKHBRIGHT fractions (Fig. 4A, BGo), 80%–90% of cells were PYLOW. In contrast, 70%–80% of PKHINTERMED cells were PYHIGH (Fig. 4C, DGo). 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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  		Figure 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 < .05). *Significantly more cells of the indicated phenotype compared wih the other three phenotypes in the population (p < .05). **Significantly more cells of the indicated phenotype than in either of the PYLOW phenotypes. Abbreviation: PBCP, peripheral blood progenitor cell (p < .05).

 
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 [41,42]. 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. 5AGo). CD34– and CD34+ fractions were cultured for 42 hours and analyzed for antigen expression and proliferative status. In the CD34– culture (Fig. 5BGo), most LIN– cells were CD34LOW (R2) or CD34– (R1). In the CD34+ culture (Fig. 5CGo), 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.

Initial CD34 Phenotype and the Onset of Cell Proliferation
Figures 3Go, 5Go, and 6Go 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 < .05; Table 3Go). By day 3, the PKHBRIGHT fraction constituted only 14% of the CD34+ culture, compared with 45% of the CD34– suspension (p < .02). More rapid outgrowth of the CD34+ population is not attributable to binding of antibody to CD34 antigen during isolation, since cell cycle parameters are unchanged by such treatment [4]. It was not determined whether the kinetic differences could be eliminated by other cytokine combinations. It was previously shown that CD34– UCB cells were slower than CD34+ cells to proliferate in a stromal system [29].

Modulation of CD34 Expression: Relation to Growth Potential
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. 6Go, 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. 3Go, 5Go), 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.

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. 7Go). Thus, neither rapid up-modulation nor down-modulation of CD34 antigen distinguished subpopulations that differed in growth capacity measured in vitro.



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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 6Go, 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.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The nature of CD34– hematopoietic cells is in dispute. Most studies have focused on the engraftment activity of CD34– cells from murine bone marrow and human UCB. Those data led many [13, 18, 21, 23, 26, 29, 43, 44] but not all [17, 45–47] researchers to conclude that CD34– cells are more primitive than CD34+ cells. Others have suggested that CD34 expression reflects either "activation" status or developmental change, or both [5, 11, 31–34, 48–50].

Analysis of human CD34– cells from fetal tissue, UCB, and bone marrow has been impeded by the generally poor growth achieved in stroma-free culture [16, 17, 19–21, 23, 25–29]. The present report describes a stroma-free, serum-free suspension culture in which CD34– PBPC generated hematopoietic cells for 8 weeks (Fig. 2Go). When CD34– and CD34+ PBPCs were compared, no significant differences in progenitor content or long-term growth in culture were observed. The prolonged and robust growth of CD34– cells observed here differs from previous reports. The difference might be due to the use of adult-mobilized PBPC, use of the CD34– subset, reliance on serum-free medium, or the combination of cytokines (see below). Interestingly, in further experiments not shown here, CD34– UCB cells grew poorly under the same conditions: cloning efficiency was low, and growth in suspension was marginal, confirming earlier work [21].

The proliferative activity of CD34– cultures cannot be ascribed to contamination by CD34+ cells. Reanalysis demonstrated the CD34– fraction contained <3% CD34+ cells. If contaminating CD34+ cells had been responsible for growth of the CD34– fraction, hematopoietic output per CD34– cell would have been just a small percent of that seen in the CD34+ fraction. That clearly was not the case. In addition, CD34+ cells appeared very rapidly in the CD34– fraction, too fast to be explained by low-level CD34+ cell contamination. Also, CD34– and CD34+ populations were clearly different in cell size and their kinetics of growth initiation (Figs. 3Go, 6Go; Table 3Go). When essentially the same cell-separation methods were applied to UCB, the resulting CD34– and CD34+ fractions differed markedly in growth characteristics (data not shown), and these are precisely the results that were expected from earlier studies. Finally, in three trials, reverse transcription-polymerase chain reaction (RT-PCR) analyses of PBPC CD34– cells did not detect CD34 mRNA.

While it has been known that CD34– marrow and UCB cells up-modulate CD34 [10, 11, 17, 18, 21, 26, 27, 30–35], many questions remain (see Introduction). For example, a link between expression of CD34 antigen and cell cycling has been proposed but has not yet been definitively shown. In fact, it was suggested that activation may differ from cell cycling [34,35]. Furthermore, stem cells’ down modulation of CD34 antigen has been reported [11, 31–35], but samples were obtained months after transplantation, making the relationship to cell cycling difficult to assess. Our finding that mobilized adult CD34– cells proliferate vigorously allowed us to study CD34– precursors in detail. In the present work, experiments showed that cultured CD34– cells upregulate CD34 antigen expression in as little as 42 hours. The rapidity of this process has not been appreciated. Whether rapid surface expression is due to a pool of cytoplasmic antigen is not known. CD34 expression increased as CD34– cells shifted from quiescence to proliferation, as measured by PKH (Fig. 3Go), PY, and Ki67 (Fig. 5Go). Cells expressing CD34 at low levels (Fig. 5BGo, region R2) showed low proliferative activity. Within the resolution of these methods, upregulation of CD34 occurred near the onset of proliferation. It seems unlikely that CD34– cells can grow while maintaining a CD34– phenotype.

With regard to CD34+ cells, CD34 antigen was lost by LIN– cells when quiescence extended beyond 2–3 days in culture (Figs. 3Go, 5Go, 6Go; Table 2Go). Loss of CD34 antigen expression was not due to maturation (the cells were uncommitted) or cell deterioration (the cells were functional [Figs. 6Go, 7Go]). We believe this is the first demonstration that primitive CD34+ cells downregulate CD34 expression in response to (or because of) extended quiescence in culture. After down-modulation of CD34 by quiescent CD34+ cells, cells retained the capacity to re-express CD34 upon initiation of growth (Figs. 6Go, 7Go). CD34+ cells retained CD34 expression in culture if they initiated growth quickly or expressed CD38 (Fig. 4AGo; see below). It is not clear whether CD34 expression is a secondary result of cells’ preparation for cycling, or whether CD34 expression is directly involved in the onset of growth. It was recently reported that quiescent mast cells upregulated CD34 as cells approached S phase [52]. Note that the relationship between CD34 antigen expression and cell proliferation differs for cultured cells and circulating cells. That is, both CD34– and CD34+ cells are quiescent in circulation. After 2 days’ cultivation, a positive correlation between CD34 expression and proliferation becomes apparent. The reason for this difference is not clear. It is possible that conditions within the circulation either do not support or actively inhibit cycling.

Further insights into CD34 expression patterns were made in Figure 4Go. When CD34+CD38– cultures were examined after 4–6 days, some quiescent cells expressed CD34 while others did not (Fig. 4A, BGo). Interestingly, the quiescent cells that expressed CD34 were CD38+, whereas those that had lost CD34 were CD38– . Although CD38 has been often considered a marker of hematopoietic differentiation, it also has been linked to regulation of cell cycle progression, growth enhancement, and activation of calcium flux [51,53]. Thus, PKHBRIGHT PYLOW cells that expressed CD34 and CD38 may have been at an early stage of activation. In some experiments, CD38 expression preceded expression of CD34 by quiescent LIN– cells (Fig. 6Go, Panel I). Together, the data suggest that expression of CD34 and CD38 by primitive cells may be early indicators of cellular activation or proliferation (or both). These findings, obtained in vitro, do not pertain to the in vivo model of CD34/CD38 reciprocity [31].

On average, cells in the CD34– population were slower to initiate growth than were CD34+ cells (Table 3Go). It is possible that mobilization activated both CD34– and CD34+ cells, but to different extents. For example, mobilization might have enhanced CD34– cells’ susceptibility to in vitro cytokine stimulation compared to non-mobilized, poorly growing CD34– cells, i.e., those from UCB. Even so, mobilized CD34– cells were slower to initiate growth than were mobilized CD34+ cells.

This report is based on the response of one specific sub-population of CD34– cells cultured in serum-free medium with a single combination of cytokines. Other media, cytokines, and subsets were examined, but little growth was obtained. For example, serum-based medium supported neither proliferation nor colony formation by CD34– PBPC, nor did other cytokine combinations such as [SCF + FL + TPO] and [SCF + TPO]. We also found that the prominent CD34– CD38+ LIN– fraction grew poorly.

In summary, both CD34– and CD34+ PBPC expressed CD34 during ex vivo proliferation. Conversely, during protracted in vitro quiescence, CD34+ cells lost CD34 antigen, and CD34– cells remained CD34– . Modulation of CD34 expression in the first several days’ culture does not affect long-term proliferative potential in vitro. These results show that determinations of CD34+ cell frequency in expansion cultures can underestimate hematopoietic content.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This research was supported by the American Red Cross.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received September 2, 2003; accepted for publication January 5, 2004.



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