Direct observation of hematopoietic progenitor chimerism in fetal freemartin cattle
© Niku et al; licensee BioMed Central Ltd. 2007
Received: 08 May 2007
Accepted: 07 November 2007
Published: 07 November 2007
Cattle twins are well known as blood chimeras. However, chimerism in the actual hematopoietic progenitor compartment has not been directly investigated. Here, we analyzed fetal liver of chimeric freemartin cattle by combining a new anti-bovine CD34 antibody and Y-chromosome specific in situ hybridization.
Bull-derived CD34+ cells were detected in the liver of the female sibling (freemartin) at 60 days gestation. The level of bull-derived CD34+ cells was lower in the freemartin than in its male siblings. Bull (Y+) and cow hematopoietic cells often occurred in separate clusters. Around clusters of Y+CD34+ cells, Y+CD34- cells were typically observed. The thymi were also strongly chimeric at 60 days of gestation.
The fetal freemartin liver contains clusters of bull-derived hematopoietic progenitors, suggesting clonal expansion and differentiation. Even the roots of the hematopoietic system in cattle twins are thus strongly chimeric from the early stages of fetal development. However, the hematopoietic seeding of fetal liver apparently started already before the onset of functional vascular anastomosis.
Cattle twins are well known as blood chimeras . Vascular anastomosis occurs in about 92% of cases, from the 10- to 15-mm crown-rump stage or 30–35 days gestation [2–5]. The blood is thus effectively mixed between the fetuses for most of the 280-day gestation. Postnatally, the twins permanently share composite blood types. This suggests that hematopoietic stem cells (HSCs) are exchanged and successfully engraft in the recipient . However, direct assessment of chimerism in the hematopoietic progenitor compartment has not been possible due to technical limitations.
Donor-derived cells can be readily identified in a cow born as a twin to a bull, as only bull cells contain a Y chromosome. Such chimeric females are usually nonfertile and are called freemartins . We have previously used genomic in situ hybridization to analyze the fates of bull-derived cells in freemartin tissues . Recently, we produced an antibody against the bovine sialomucin CD34 for identification of cattle hematopoietic progenitors . Here, we combine these tools to directly observe bull-derived hematopoietic progenitors in the liver of an early fetal freemartin.
Results and Discussion
Quantification of bull-derived CD34+ cells in the livers of bovine triplet fetuses. Proportions of Y-chromosome positive (Y+) cells among liver CD34+ cells in the 60-day fetuses are shown, as detected by in situ hybridization. fm = freemartin, n = number of image fields analyzed.
CD34+ cells total
% Y+ (mean ± SD)
21.8 ± 4.1
<0.01 (bull 1)
<0.001 (bull 2)
30.9 ± 5.4
>0.05 (bull 2)
38.4 ± 6.7
>0.05 (bull 1)
We have previously shown that the Y-ISH method applied here is extremely specific and sufficiently sensitive . No false positives were detected in more than 1000 samples of various tissues from normal females (not shown and Fig. 1c). A proportion of bull-derived cells are not detected, as the Y chromosome may be excluded from the section. In normal bull tissues, generally 40–70% of cells are labelled depending on the cell type (here, see Fig. 1a).
CD34 is a transmembrane glycoprotein commonly used as a marker for enrichment of human hematopoietic stem cells . While the CD34+fraction is heterogenous and contains also some committed precursors , CD34 remains as one of the best known markers for primitive hematopoietic cells . Recently, bovine CD34+ cells were shown to be enriched for hematopoietic progenitors as measured by BFU-E (burst-forming unit – erythroid) and CFU-GM (colony-forming unit – granulocyte-monocyte) readouts . The polyclonal antibody used in our study specifically recognizes bovine CD34 . In addition, it labels erythroid cells. These were excluded from the analysis by pseudoperoxidase staining, as described in Methods.
This is the first study to directly demonstrate bull-derived hematopoietic progenitors in fetal freemartin cattle. The fetal liver contains clusters of bull-derived cells, suggesting clonal expansion and differentiation. The lower level of bull-derived hematopoietic progenitors in the freemartin vs. her bull siblings suggest that the seeding of fetal liver starts already before the onset of vascular anastomosis. The level of chimerism in the circulating migratory hematopoietic progenitors thus only partially determines the level of donor-derived fetal hematopoiesis. Despite this, the freemartin hematopoietic system is strongly chimeric already from early stages of fetal development.
Fetuses were obtained at an abattoir from a superovulated cow. Superovulatory treatment was initiated 11 days after estrus. The cow was administered intramuscularly (i.m.) total of 360 mg of NIH-FSH-P1 (FolltropinR-V, Bioniche, Ireland) divided in decreasing doses at 12 h intervals over four days. Luteolysis was induced with an i.m. injection of 0.15 mg of dexcloprostenol (GenestranR, 0.075 mg/ml, Vetcare, Salo, Finland) 60 h after the initiation of the superovulatory treatment. The cow was artificially inseminated 48 and 60 h after the dexcloprostenol administration.
Animal experiments were approved by the local animal welfare authorities.
Combined in situ hybridization and immunofluorescence
Combined Y-chromosome specific in situ hybridization and CD34 immunostaining were performed essentially as described previously .
Here, erythroid cells were first stained by their pseudoperoxidase activity using tyramide amplification and the diaminobenzidine (DAB) substrate (Vector Laboratories), as described previously . Genomic in situ hybridization was then performed, using microwave heating, protease treatment, and a Y-chromosome specific oligonucleotide probe carrying a digoxigenin label. The hybridized probes were detected by alkaline phosphatase-conjugated anti-DIG-Fab fragments (Roche) and visualized using the NBT/BCIP chromogen (Roche). CD34 was then detected by immunofluorescence. The sections were subjected to heat-induced antigen retrieval and a protease treatment, incubated overnight in the polyclonal anti-bovine CD34 antibody, and then incubated in Alexa546-conjugated anti-rabbit Ig antibody (Invitrogen). Finally, autofluorescence was quenched by Sudan Black B (Merck).
The sections were examined and photographed using a Leica DM4000 epifluorescence microscope equipped with an Olympus DP70 camera. Merged images were prepared in Adobe Photoshop.
Y-chromosome positive and negative CD34+ cells in fetal livers were counted in merged images of double-stained tissue sections, using the Cell^P image analysis software (Olympus). More than 500 CD34+ cells were counted in each animal. Statistical significance was evaluated using independent samples t test.
Y-chromosome positive cells were also counted in fetal thymi. Numbers of Y-chromosome negative cells were estimated based on cell densities in hematoxylin-eosin stained serial sections of the same tissues. In total, more than 2000 thymic cells per animal were included in the analysis.
BCIP, 5-bromo-4-chloro-3-indolyl phosphate; CD, cluster of differentiation; DIG, digoxigenin; FSH, follicle stimulating hormone; NBT, nitro blue tetrazolium; SD, standard deviation
We thank Kirsi Lahti and Tuire Pankasalo for expert technical assistance. The study was funded by Academy of Finland (210978/10.11.2004 to AI), The University's research funds (914/51/2006 to AI), Mercedes Zachariassen's Foundation (to AI) and Finnish Veterinary Foundation (to AI). All authors are employed by the University of Helsinki.
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