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Keywords:

  • Fetal blood;
  • Umbilical cord blood;
  • CD34;
  • Stem cell

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

The objective of this study was to compare the expression of primitive cell-surface antigens on CD34+ cells from early in gestation to those from term gestations. Fetal blood samples were obtained from 10 early gestation (21.0 ± 0.8 [SE] weeks) and 12 term gestation (39.3 ± 0.4 weeks) fetuses. The mononuclear cell population was separated by red cell lysis. Two-color flow cytometry was used to assess cell surface antigen coexpression of CD34 with CD33, CD38, and HLA-DR as well as staining by a cocktail of monoclonal antibodies for lineage-associated (Lin) antigens (CD2, CD10, CD11b, CD19, CD20, CD33, CD36, 7B9, and Glycophorin-A). The frequency of CD34+ cells (5.5 ± 0.9 versus 1.5 ± 0.2, p < 0.001) was significantly higher in the early gestational age group. Within the CD34+ population, the frequency of CD34+/CD38 cells (81.8 ± 9.9 versus 51.3 ± 7.7, p = 0.02) and CD34+/DR cells (15.3 ± 7.4 versus 8.2 ± 2.7, p = 0.05) was also higher in the early gestational age group. In contrast, CD34+/CD33 (51.8 ± 10.1 versus 83.0 ± 6.1, p = 0.02) and CD34+/Lin cells (15.9 ± 7.0 versus 51.8 ± 6.9, p < 0.01) were higher in the term gestation group. The high percentage of CD34+, CD34+/CD38, and CD34+/DR cells supports our hypothesis that early gestational age fetal blood has a higher frequency of primitive hematopoietic progenitor/stem cells than does umbilical cord blood at term. This suggests that hematopoietic progenitor/stem cells in early fetal blood may be a desirable target for in utero gene therapy. However, further studies to characterize the functional properties of CD34+ cell subsets at different stages of fetal development will be necessary to determine the appropriateness of targeting fetal hematopoietic cells for in utero gene therapy. The higher frequency of CD34+/CD33 and CD34+/Lin cells from term gestational age fetuses was unexpected, and the significance of this finding is unclear at this time.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Human umbilical cord blood obtained at delivery has been shown to be a potential source of hematopoietic stem cells (HSC) for postnatal transplantation [1-4]. Umbilical cord blood at the time of delivery contains a high frequency of progenitor cells with a frequency of CD34+ cells comparable to mobilized adult peripheral blood, although less than adult bone marrow [5-9]. Early in gestation, the frequency of CD34+ progenitors is equal to or greater than that of bone marrow [10] and the number of progenitor cells appears to be increased [7, 9, 11]. During maturation of hematopoietic cells, there is an orderly progression of cell-surface antigens that are expressed or lost. The characterization of the cells by their antigen expression during hematopoiesis is important in identifying cells within the continuum of development from stem cell to progenitor cell and finally to mature effector cell [12-14]. The CD34 antigen is expressed by virtually all human hematopoietic progenitors and their precursors, including most, if not all, stem cells [15, 16]. As cells differentiate and become lineage-committed, subsets of CD34+ cells can be identified by the presence of specific cell-surface antigens, while the absence of such antigens identifies cells as immature and pre-lineage-committed [17-19]. Expression of antigens associated with specific stages and lineages of hematopoietic differentiation has been used to characterize HSC in adult bone marrow and peripheral blood, term umbilical cord blood, fetal liver, and fetal bone marrow [5, 6, 12]. The purpose of this study was twofold: first, to compare the frequencies of CD34+ cells co-expressing CD33, CD38, HLA-DR, or a cocktail of lineage-related antigens, by early (17-24 weeks' gestation) and term (38-41 weeks' gestation) human fetal blood cells using two-color flow cytometry, and second, to evaluate the growth and proliferation potential of early and term gestation fetal blood by colony-forming assays.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Cord Blood Samples

Fetal blood samples were obtained from excess umbilical cord blood from term gestations and at the time of elective terminations of pregnancy. Fetal blood was placed into EDTA tubes immediately after it was drawn. Red cells were lysed with an ammonium chloride/EDTA lysis buffer [20], using either the entire blood volume for small samples (n = 3, all from preterm samples) or buffy coat cells for large volumes (n = 19, 12 from term samples, and 7 from preterm). The resulting cells were washed twice in cold phosphate-buffered saline (PBS) plus 2% heat-inactivated human AB serum ([HABS]; Gemini Bio-Products; Calabasas, CA), counted, and cryopreserved at –70°C in 90% fetal bovine serum ([FBS]; Hyclone Laboratories, Inc.; Logan, UT) plus 10% dimethylsulfoxide ([DMSO]; Sigma; St. Louis, MO). Samples were defined as “early gestational age” if they were from fetuses that were less than 24 weeks' gestation (mean gestational age 21 ± 2.4 weeks, n = 10) and “term gestational age” if they were between 38-41 weeks' gestation (mean gestational age 39.3 ± 1.5 weeks, n = 12). This project, including the collection of fetal blood, was approved by the University of Washington Human Subjects Committee.

Antibodies

The monoclonal antibodies used in this study included IgG antibodies to CD33 (P67-6, isotype IgG1; Irwin Bernstein, Fred Hutchinson Cancer Research Center [FHCRC]; Seattle, WA), CD38 (OKT10, IgG1; Coulter Corporation; Miami, FL), HLA-DR (4.1, IgG1; Paul Martin and John Hanson, FHCRC), and lineage associated antigens (Lin) comprised of CD2 (9.6, IgG2a), CD10 (24.1, IgG3), CD11b (60.1, IgG2a), from Paul Martin and John Hanson, FHCRC; CD19 (HD37, IgG1) from Ed Clark, University of Washington; CD20 (B1, IgG2a), CD33 (P67-6), CD36 (F13, IgG3) from Irwin Bernstein, FHCRC [21]; R10 (glycophorin A) and 7B9 (myeloid-associated antigen, IgG1) [22]. Antibodies 31A (IgG1) [23], 1A14 (IgG2a) [24], 5.31 (IgG3; John Hanson, FHCRC) against mouse Thy 1.1 antigen served as isotype-specific control antibodies with irrelevant specificity. IgM antibodies used were CD34 (12.8; FHCRC) and the isotype control H12C12 (anti-mouse Thy 1.2) [24]. The above antibodies were purified from ascites fluid or used as a dilution of ascites. The IgG antibodies were used at a concentration of 10 μg/ml for purified antibodies or a dilution of 1:1000 for those obtained from ascites. The IgM antibodies were used at a concentration of 20 μg/ml or a 1:500 dilution of ascitic fluid. FITC-conjugated isotype-specific goat-antimouse IgG1, IgG2a, and IgG3 (PharMingen; San Diego, CA) and phycoerythrin (PE) goat-antimouse IgM (AMAC Inc.; Westbrook, ME), each at 1:40 dilution were used to stain labeled cells for flow cytometry.

Cell Staining and Flow Cytometry (FACS)

Frozen blood samples were thawed in a 37°C water bath and resuspended in RPMI plus 10% FBS and 100 U/ml DNAse (Sigma), washed twice with cold PBS-2% HABS and 100 U/ml DNAse. The viability of the thawed cells was 80%-99%, as determined by trypan blue dye exclusion. Cells were resuspended in PBS-2% HABS for staining with monoclonal antibodies. Cells were aliquoted into 96-well V-bottom sterile microtiter plates and centrifuged at 1,200 RPM for four min. The cells were resuspended with the appropriate monoclonal antibodies and incubated at 4°C for 20 min. The cells were then washed twice with PBS-2% HABS and incubated with the fluorescein isothiocyanate-G (FITC-G) and phycoerythrin-M (PE-M)-conjugated second-step antibodies at 4°C for 15 min. Propidium iodide (Sigma) at 8 μg/ml was added to each well to stain for viability and incubated at 4°C for five min. The cells were then washed twice with PBS-2% HABS prior to analysis by flow cytometry.

Flow analysis was done on a Becton-Dickinson FACSCAN® using B-D CellQuest software (Becton-Dickinson Immunocytometry Systems; San Jose, CA). Maximum numbers of events were collected due to the low numbers of CD34+ cells anticipated per sample. Events were collected with an open gate and later gated for forward and 90-degree light scatter to isolate lymphocyte and mononuclear sized cells, then gated for propidium iodide staining to remove nonviable cells. There were 85%-89% viable cells in the selected lymphocyte/monocyte window. CD34+ cells were selected by gating on a discrete population of brightly stained 34+ cells that had fluorescence intensity greater than 99% of isotype control. CD34+ cells were considered positively stained for FITC-IgG (CD38, HLA-DR, and lineage-associated antigens) if they displayed a fluorescence intensity that was greater than 95% of the isotype control. Samples with fewer than 100 CD34+ events were not included in the analysis.

In Vitro Colony Assays

Colony assays were performed using a two-layer agar method with 25% serum, as previously described [25]. Briefly, the bottom layer consisted of 0.5 ml 2× culture media plus 0.5 ml of 1% Seaplaque agar (FMC Bio-Products; Rockland ME) and the following hematopoietic growth factors: interleukin 3 (IL-3), IL-6, stem cell factor (SCF), and GM-CSF, each at 100 ng/ml, and erythropoietin (Epo) at 4 U/ml (provided by Dr. Ian McNiece, Amgen Inc.; Thousand Oaks, CA). The top layer consisted of 0.25 ml of 2× culture media plus 0.25 ml of 0.6% Seaplaque agar and cells. Cells were plated at a concentration of 50,000/plate for term samples and 10,000/plate for early samples. The culture medium was made as a 2× solution consisting of 50% fetal calf serum (Hyclone), 40% 3× alpha-minimal essential medium ([α-MEM] GIBCO; Grand Island, NY), 0.2% (500 mg/25 ml) bovine serum albumin (fraction V, Sigma) freshly dissolved in 3× α-MEM, 2% glutamine and 100 μg/ml gentamicin sulfate. Cultures were incubated at 37°C in 5% CO2 for 14 days and scored as BFU-E and granulocyte-macrophage colony-forming units (CFU-GM) based on colony morphology.

Statistical Evaluation

Differences between the two groups were determined by Student's t-testing.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Coexpression of Lineage-Associated Differentiation Antigens by CD34+ Cells From Fetal Blood

Mononuclear cells from early gestation (21.0 ± 2.4 weeks, n = 12) and term (39.3 ± 1.5 weeks, n = 12) were analyzed by flow cytometry for expression of CD34 as well as coexpression of CD33, CD38, HLA-DR, and reactivity with a pool of lineage-associated antigens (Lin), CD2, CD10, CD11b, CD20, CD33, CD36, glycophorin A, and 7B9. Early-gestation fetal blood contained a higher frequency of CD34+ cells than term-gestation umbilical cord blood (7.1 ± 1.1% versus 1.5 ± 0.2%, p < 0.001, Fig. 1). We then determined whether the proportions of CD34+ cells with phenotypes associated with differing levels of maturity in adult marrow [17-19] were influenced by gestational age. The frequency of CD34+ cells that are CD38 was significantly greater in early gestation than in term gestation (81.8 ± 9.9% versus 51.3 ± 7.7%, p = 0.02, Fig. 2). Similarly, the proportion of CD34+ cells that were HLA-DR was also higher in early fetal blood than in term (15.3 ± 7.4% versus 8.0 ± 2.7%, p = 0.05, Fig. 2). In contrast, the frequency of CD34+ cells that were also lacking expression of CD33 (57.8 ± 10.1% versus 83.0 ± 6.1%, p = 0.02) or lineage-associated antigens (14.9 ± 7.0% versus 51.8 ± 6.9%, p = 0.01) was lower in cells from early-gestation fetal blood than from term-gestation fetal blood (Fig. 2).

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Figure Figure 1.. The frequency of CD34+cells (as a percentage) within the mononuclear cell population from early (17-24 weeks) and term (37-41 weeks) gestation fetal blood.

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Figure Figure 2.. The frequency of CD34+cells that were not coexpressing CD38, HLA-DR, CD33, and lineage-associated antigens CD2 (9.6), CD10 (24.1), CD11b (60.1), CD19 (HD37), CD20 (B1), CD33 (P67-6), CD36 (F13), glycophorin A (R10), and myeloid-associated antigen (7B9).

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Progenitor Cells in Fetal Blood

Unfractionated fetal blood cells from early and term gestation were cultured in semisolid medium to compare the frequency of progenitors (CFU-GM and BFU-E). Early fetal blood contained significantly more colony-forming cells (CFCs) (560 ± 116/50,000 cells cultured, n = 8) than term umbilical cord blood (64 ± 14/50,000 cells cultured, n = 10; p < 0.001). This increased frequency was due to a greater number of both CFU-GM and BFU-E. Early fetal blood samples produced 419 ± 107 BFU-E and 141 ± 15 CFU-GM per 50,000 cells cultured, compared to 32 ± 14 (p < 0.001) and 32 ± 5 (p = 0.002) respectively from term fetal blood.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

The goal of this study was to further characterize fetal hematopoiesis by examining the expression of cell-surface antigens associated with progenitor cells and their precursors in fetal blood from early and term gestation. We found that the frequency of hematopoietic progenitor cells, defined by both in vitro colony assays and expression of CD34, is markedly elevated in early second trimester fetal blood (17-24 weeks) compared to term gestation fetal blood (37-41 weeks) (Fig. 3). This observation is consistent with other studies that used CFC assays [7, 11, 26] and fetal blood [10, 27], which have shown that circulating progenitors and CD34+ cells decrease with increasing gestational age. Clapp et al. [26] assayed CFC from preterm cord blood (25 to 36 weeks' gestation) and term cord blood (38 to 42 weeks' gestation) and demonstrated an age-dependent decrease in circulating hematopoietic progenitors. Jones et al. [11] have shown that the frequency of BFU-E in second trimester fetal blood is higher than that in term blood. Similarly, studies by Andreux et al. [7] demonstrated that the frequency of BFU-E from second-trimester (19-30 weeks) fetal blood was three times higher than from umbilical cord blood obtained from full-term gestations. Of particular interest, BFU-E decreased to a much greater extent with advancing gestational age than did CFU-GM. This observation likely reflects the large fetal requirement for erythroid-committed progenitors necessary for red cell production during fetal development (an increase of approximately 2%/day) [28]. However, little is known about the expression of cell-surface antigens by circulating progenitors early in gestation which may distinguish phenotypically immature cells from more mature progenitor cells.

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Figure Figure 3.. In vitro colony-forming assays, BFU-E and granulocyte-macrophage colony-forming units (CFU-GM) from early (17-24 weeks) and term (37-41 weeks) gestation fetal blood.

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When we examined the coexpression of CD38 and HLA-DR by CD34+ cells as surrogate measures for the level of maturation, we noted that fetal blood from early in gestation had a significantly higher proportion of CD34+ cells that were both CD38 and HLA-DR. Surprisingly, in contrast to the HLA-DR and CD38 data, the proportion of CD34+ expressing CD33 as well as lineage antigens (Lin) was higher in early fetal blood. Cells capable of extensive self-renewal and/or multilineage differentiation have been found within the CD33, CD38 and HLA-DR subpopulations of CD34+ cells [12, 24, 29]. Although term umbilical cord blood has a lower frequency of CD34+ cells than adult bone marrow, term umbilical cord blood is thought to have a higher proportion of functionally immature progenitors [6] and a higher proportion of phenotypically more primitive CD34+ cells defined by lack of expression of CD38 and CD33 [8]. Functional studies of phenotypically defined cells from umbilical cord blood and adult bone marrow suggest that CD34+CD38 cells have a higher cloning efficiency, proliferate more rapidly in the presence of cytokines, and generate more progenitors than do CD34+CD38 cells from adult bone marrow [6, 13]. The higher frequencies of CD34+CD38 and CD34+HLA-DR cells from early gestational age fetal blood in our studies suggest that there are significantly more of these immature cells in the circulation early in gestation than in the fetus at term. This finding is consistent with those of Thilaganathan et al. who also noted a higher frequency of CD34+/HLA-DR and CD34+/CD38 cells in early fetal blood relative to term gestation [27]. Interestingly, the frequency of CD34+CD33 and CD34+Lin cells was lower early in gestation than at term. This finding was unexpected based on the high frequency of CD34+CD38 and CD34+HLA-DR. Thilaganathan et al. evaluated the frequency of coexpression of CD33 on CD34+ cells across gestation. They noted that the frequency of CD34+CD33 was low early in gestation (18% at 13-15 weeks). Therefore, based on our results and those of Thilaganathan et al., there appears to be a progressive increase in the frequency of CD34+CD33 cells with advancing gestation. This, along with our finding of higher frequencies of CD34+Lin cells at term, raises the possibility that expression of some cell surface lineage-associated antigens, used to identify the phenotypic or functional maturity of postnatal CD34+ cells, may be less useful in defining particular points during the continuum of fetal hematopoietic development. Further studies to characterize the functional properties of CD34+ cell subsets at different stages of fetal development will be necessary to understand the changes in circulating stem and progenitor cells and their appropriateness as targets for in utero gene therapy.

In summary, we noted that the frequency of CD34+ cells as well as CD34+ cells that were also CD38 and HLA-DR, was higher in early fetal blood. These data suggest that CD34+ cells from early in gestation are functionally less mature than CD34+ cells obtained at term and that hematopoietic progenitors from early in gestation may be a more desirable target for hematopoietic gene therapy and transplantation. We also noted that the frequency of cells that were CD34+/CD33+ and CD34+/Lin+ was higher in early fetal blood. The high lineage antigen positivity may be related to large numbers of erythroid-committed progenitors that are necessary for the rapidly expanding fetal hematopoietic environment. Further studies characterizing the functional properties of subsets of CD34+ cells will be needed to help elucidate these findings.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References
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