Limited Contribution of Circulating Cells to the Development and Maintenance of Nonhematopoietic Bovine Tissues
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《干细胞学杂志》
a Division of Anatomy and
b Division of Biochemistry, Department of Basic Veterinary Sciences, University of Helsinki, Helsinki, Finland
Key Words. Stem cells ? Blood circulation ? Regeneration ? Chimera ? Freemartinism ? Y chromosome ? In situ hybridization
Antti Iivanainen, D.V.M., Ph.D., Department of Basic Veterinary Sciences, FIN-00014 University of Helsinki, Helsinki, Finland. Telephone 358-9-191-49544; Fax: 358-9-191-49799; e-mail: Antti.Iivanainen@helsinki.fi
ABSTRACT
The differentiation potential of stem cells (SCs) in adult mammals is currently a topic of intense debate. Many recent reports have challenged the traditional dogma of strictly restricted differentiation options of adult cells. Bone marrow (BM) cells, hematopoietic stem cells (HSCs), or mesenchymal stem cells (MSCs) have been shown to differentiate into cells of the skeletal muscle , liver , epithelia of skin, lung, and gastrointestinal tract and even to cells of the central nervous system (CNS) . However, the physiological significance of the findings is uncertain. The cell culture techniques and transplantation methods applied are likely to induce changes both in the cells and recipient tissues . Also, heterogeneity of the transplanted cell populations , potential cell fusion events , and problems in reproducing some of the results complicate interpretation of transdifferentiation experiments.
Circulating cells derived from BM or other sources may have a role in tissue repair . A traditional model of regenerating tissue is a subcutaneously implanted cellulose viscose sponge . The sponge is invaded by irregular, richly vascularized granulation tissue similar to that forming in a healing wound. Granulocytes and macrophages first enter the sponge, followed by fibroblasts, and a rapidly increasing collagen synthesis and angiogenesis in a few days . The origin of the granulation tissue producing cells is unclear, with several contradictory studies supporting either a local or systemic source .
We have used freemartin cattle to monitor the contribution of prenatally circulating cells to the development, physiological maintenance, and repair of bovine tissues. Freemartin is a female calf born as a twin to a bull. About 90% of cattle twins are chimeric due to placental anastomoses that form early in the development and permit the exchange of blood and other cells between the fetuses . The twins permanently share identical composite blood types and are mutually tolerant to allografts from each other . The proportions of donor-derived nucleated cells in blood and BM of different animals are randomly distributed between less than 5% and more than 95% . The freemartins develop normally, apart from the inner sex organs, which are masculinized to a variable degree probably due to hormonal influences . Donor-derived cells in freemartin tissues can be readily identified by their Y chromosomes. Thus, the freemartin is a suitable model for studies of SC transfer in an unmanipulated large mammal, and also unique in allowing the investigation of prenatal events in a chimeric animal.
Freemartin tissues have formerly been analyzed by cytogenetic techniques , but little is known about the contribution of donor-derived cells to the development, growth, and cell dynamics of the non-hematopoietic organs. We have previously characterized the freemartin immune system by Y-chromosome-targeted in situ hybridization (Y-ISH) , which allows the identification of bull-derived cells in intact tissue sections. Y-ISH can be combined with immunohistochemistry or other staining methods to investigate the phenotypes of cells. In this paper, we describe an extensive mapping of freemartin tissues using Y-ISH combined with CD45 immunohistochemistry and mistletoe lectin I (ML-I) histochemistry to exclude cells of the hematopoietic lineage. CD45 is a cell surface antigen exclusively expressed by all nucleated cells of the hematopoietic lineage . ML-I labels bovine microglia .
MATERIALS AND METHODS
We first performed a wide Y-ISH screening of tissues from 12 freemartins (ages: 20 days to 20 months). Samples from 20 to 74 different body sites per animal were analyzed. Y-chromosome positive (Y+) bull-derived cells were detected in all tissues examined in all 12 animals. The highest frequencies of Y+ cells were found in blood, BM, and lymphatic tissues. These tissues also exhibited considerable variation between individual animals. Among blood mononuclear cells, the proportion of Y+ cells varied from approximately 10% to 90%. In other organs, the variation was less pronounced.
The distribution of Y+ cells suggested that a significant proportion of them were infiltrating leukocytes, which often are difficult to distinguish reliably from parenchymal cells in a standard histological staining. To label the leukocytes residing in the tissues, we combined Y-ISH and CD45 immunohistochemistry or, in the case of CNS, ML-I histochemistry for bovine microglial cells . We re-examined all major tissue types and derivatives of all embryonic layers. The double staining confirmed that most Y+ cells indeed were of the hematopoietic lineage (Fig. 1A–1G). CD45/ML-I-Y+ cells were identified in most tissues, but in 11 of 12 calves (208 succesfully double-stained sections from 14 different tissues) they were mostly sporadic. No groups of CD45/ML-I-Y+ cells indicating local clonal expansion were seen in these calves. However, one of the calves (FM10) was a striking exception, with high numbers of CD45/ML-I-Y+ cells in several tissues. This case is discussed more thoroughly below.
Figure 1. Bull-derived (Y+) cells in freemartin tissues. Y-chromosome targeted in situ hybridization combined with anti-leukocyte staining (MLI histochemistry for brain, anti-CD45 immunohistochemistry for other tissues). A-G) physiological freemartin tissues: A) cerebral cortex; B) renal cortex; C) liver; D) mammary gland, duct epithelium; E) mammary gland, connective tissue around ducts; F) skeletal muscle; G) mucosa of the small intestine; H) intestinal mucosa from FM10, showing exceptionally strong donor contribution to intestinal epithelium; I) granulation tissue in a cellulose viscose sponge, incubation time 4 weeks. White arrowheads: CD45/ML-I-Y+ cells. Black arrowheads: CD45/ML-I+Y+ cells. Asterisk: a chimeric foreign body giant cell. Bars: 25 μm.
The donor contribution to the nonhematopoietic cells in various tissues was estimated by counting the proportion of CD45/ML-I-Y+ cells in 4 to 5 calves per tissue (including the exceptional case). Altogether, several tissues were analyzed from all 12 freemartins. Apart from FM10, the calves did not differ markedly in the relative frequency of CD45/ML-I-Y+ cells, which constituted less than 1% of all CD45/ML-I- cells in most tissues investigated (Fig. 2). There was no obvious correlation between the frequency of Y+ cells in blood and in nonhematopoietic tissues, or between different nonhematopoietic tissues in an animal. Donor-derived nonhematopoietic cells were markedly rare in the ectoderm-derived epidermis and CNS. Otherwise, no consistent differences between derivatives of different embryonic layers were seen. In 11 of 12 calves, highest numbers of CD45/ML-I-Y+ cells were detected in the connective tissue around ducts of mammary glands (0.94 ± 0.67%), liver (0.85 ± 0.41%, primarily not hepatocytes), and skeletal muscle (0.83 ± 0.26%). In cardiac and smooth muscle, renal epithelia, and epidermis the average frequency of CD45-Y+ cells was 0.1% or less (in epidermis the interdigitating Langerhans cells make the identification of CD45- cells difficult). In the brain, very few ML-I- donor-derived cells were found, although donor contribution to the microglia was in most cases substantial (we note that in addition to microglial cells, the lectin labels most endothelia). In the cerebral cortex and the underlying white matter, 0.0020 ± 0.0023% of ML-I- cells were Y+. In the hippocampus and the OB, two regions involved in the generation of new neurons in adult mammals , no ML-I-Y+ cells were detected in approximately 2 x 105 cells screened in double-stained sections of each tissue. No Y+ cells with clearly neuronal morphology were seen in about 720 successfully hybridized sections from freemartin CNS, representing approximately 28 x 106 total cells and including samples from cerebral cortex, cerebellum, brain stem, corpus callosum, hippocampus, OB, thalamus, ventricular walls, hypophysis, and spinal cord.
Figure 2. Proportions of bull-derived (Y+) cells in all nonhematopoietic (CD45/ML-I-) cells in 11 majority-type freemartins. Each tissue was analyzed from four majority-type calves (not the same calves for different tissues), with the exception of the mammary gland (three samples containing ducts available), and the regenerating tissue in implants (one sample per time point). Numbers were corrected with counts obtained from similar bull tissues as described in Materials and Methods. EP = epithelium, CT = connective tissue, MU = muscle tissue, NE = nervous tissue. For each tissue, individual samples are arranged in order of increasing animal age.
In FM10, the distribution of CD45/ML-I-Y+ cells differed dramatically from the rest of the calves. Higher numbers of CD45-Y+ cells were found in the epithelium of small intestine (19%, Fig. 1H), connective tissue of mammary gland (15%), liver (4.3%), epithelium of mammary gland (4.0%), skeletal muscle (2.3%), cardiac muscle (1.0%), and smooth muscle (0.5%), whereas in brain and in renal epithelia the frequency did not differ notably from the other calves. In the villi of the intestinal mucosa, strands of CD45-Y+ cells were seen (Fig. 1H). The hematopoietic system of FM10 differed from the majority of calves in that the donor contribution to B cells was clearly weaker than to the total leukocyte pool (not shown).
To investigate the role of donor-derived cells in tissue regeneration, we implanted cellulose viscose sponges in the subcutis of a 70-day-old freemartin and stained the invading granulation tissue for Y-chromosome and CD45. The regenerating tissue contained relatively high numbers of CD45-Y+ cells, with increasing numbers within the incubation period of 4 weeks (Figs. 1I, 2). At 4 weeks, the frequency of CD45-Y+ cells in the regenerating tissue was significantly higher than in most other nonhematopoietic tissues of the same animal (p = 0.12 for liver, p < 0.02 for any other tissue investigated). The frequencies of CD45/ML-I-Y+ cells in the tissues of the recipient did not differ markedly from those in the 10 other calves of the majority type. No clear Y+ cell clusters indicating local expansion were seen.
DISCUSSION
We thank M. Georges (University of Liège, Belgium) for the btDYZ clone, U. Pfüller (Witten/Herdecke University, Germany) for the biotinylated mistletoe lectin I, staff at the Saari unit (Dept. Clinical Veterinary Medicine, University of Helsinki) for providing blood samples of potential freemartins, K. Lahti, T. Pankasalo, H. Valtonen, and A. Koivunen for expert technical assistance, and L.C. Andersson and L.A. Lindberg for valuable comments about the work.
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b Division of Biochemistry, Department of Basic Veterinary Sciences, University of Helsinki, Helsinki, Finland
Key Words. Stem cells ? Blood circulation ? Regeneration ? Chimera ? Freemartinism ? Y chromosome ? In situ hybridization
Antti Iivanainen, D.V.M., Ph.D., Department of Basic Veterinary Sciences, FIN-00014 University of Helsinki, Helsinki, Finland. Telephone 358-9-191-49544; Fax: 358-9-191-49799; e-mail: Antti.Iivanainen@helsinki.fi
ABSTRACT
The differentiation potential of stem cells (SCs) in adult mammals is currently a topic of intense debate. Many recent reports have challenged the traditional dogma of strictly restricted differentiation options of adult cells. Bone marrow (BM) cells, hematopoietic stem cells (HSCs), or mesenchymal stem cells (MSCs) have been shown to differentiate into cells of the skeletal muscle , liver , epithelia of skin, lung, and gastrointestinal tract and even to cells of the central nervous system (CNS) . However, the physiological significance of the findings is uncertain. The cell culture techniques and transplantation methods applied are likely to induce changes both in the cells and recipient tissues . Also, heterogeneity of the transplanted cell populations , potential cell fusion events , and problems in reproducing some of the results complicate interpretation of transdifferentiation experiments.
Circulating cells derived from BM or other sources may have a role in tissue repair . A traditional model of regenerating tissue is a subcutaneously implanted cellulose viscose sponge . The sponge is invaded by irregular, richly vascularized granulation tissue similar to that forming in a healing wound. Granulocytes and macrophages first enter the sponge, followed by fibroblasts, and a rapidly increasing collagen synthesis and angiogenesis in a few days . The origin of the granulation tissue producing cells is unclear, with several contradictory studies supporting either a local or systemic source .
We have used freemartin cattle to monitor the contribution of prenatally circulating cells to the development, physiological maintenance, and repair of bovine tissues. Freemartin is a female calf born as a twin to a bull. About 90% of cattle twins are chimeric due to placental anastomoses that form early in the development and permit the exchange of blood and other cells between the fetuses . The twins permanently share identical composite blood types and are mutually tolerant to allografts from each other . The proportions of donor-derived nucleated cells in blood and BM of different animals are randomly distributed between less than 5% and more than 95% . The freemartins develop normally, apart from the inner sex organs, which are masculinized to a variable degree probably due to hormonal influences . Donor-derived cells in freemartin tissues can be readily identified by their Y chromosomes. Thus, the freemartin is a suitable model for studies of SC transfer in an unmanipulated large mammal, and also unique in allowing the investigation of prenatal events in a chimeric animal.
Freemartin tissues have formerly been analyzed by cytogenetic techniques , but little is known about the contribution of donor-derived cells to the development, growth, and cell dynamics of the non-hematopoietic organs. We have previously characterized the freemartin immune system by Y-chromosome-targeted in situ hybridization (Y-ISH) , which allows the identification of bull-derived cells in intact tissue sections. Y-ISH can be combined with immunohistochemistry or other staining methods to investigate the phenotypes of cells. In this paper, we describe an extensive mapping of freemartin tissues using Y-ISH combined with CD45 immunohistochemistry and mistletoe lectin I (ML-I) histochemistry to exclude cells of the hematopoietic lineage. CD45 is a cell surface antigen exclusively expressed by all nucleated cells of the hematopoietic lineage . ML-I labels bovine microglia .
MATERIALS AND METHODS
We first performed a wide Y-ISH screening of tissues from 12 freemartins (ages: 20 days to 20 months). Samples from 20 to 74 different body sites per animal were analyzed. Y-chromosome positive (Y+) bull-derived cells were detected in all tissues examined in all 12 animals. The highest frequencies of Y+ cells were found in blood, BM, and lymphatic tissues. These tissues also exhibited considerable variation between individual animals. Among blood mononuclear cells, the proportion of Y+ cells varied from approximately 10% to 90%. In other organs, the variation was less pronounced.
The distribution of Y+ cells suggested that a significant proportion of them were infiltrating leukocytes, which often are difficult to distinguish reliably from parenchymal cells in a standard histological staining. To label the leukocytes residing in the tissues, we combined Y-ISH and CD45 immunohistochemistry or, in the case of CNS, ML-I histochemistry for bovine microglial cells . We re-examined all major tissue types and derivatives of all embryonic layers. The double staining confirmed that most Y+ cells indeed were of the hematopoietic lineage (Fig. 1A–1G). CD45/ML-I-Y+ cells were identified in most tissues, but in 11 of 12 calves (208 succesfully double-stained sections from 14 different tissues) they were mostly sporadic. No groups of CD45/ML-I-Y+ cells indicating local clonal expansion were seen in these calves. However, one of the calves (FM10) was a striking exception, with high numbers of CD45/ML-I-Y+ cells in several tissues. This case is discussed more thoroughly below.
Figure 1. Bull-derived (Y+) cells in freemartin tissues. Y-chromosome targeted in situ hybridization combined with anti-leukocyte staining (MLI histochemistry for brain, anti-CD45 immunohistochemistry for other tissues). A-G) physiological freemartin tissues: A) cerebral cortex; B) renal cortex; C) liver; D) mammary gland, duct epithelium; E) mammary gland, connective tissue around ducts; F) skeletal muscle; G) mucosa of the small intestine; H) intestinal mucosa from FM10, showing exceptionally strong donor contribution to intestinal epithelium; I) granulation tissue in a cellulose viscose sponge, incubation time 4 weeks. White arrowheads: CD45/ML-I-Y+ cells. Black arrowheads: CD45/ML-I+Y+ cells. Asterisk: a chimeric foreign body giant cell. Bars: 25 μm.
The donor contribution to the nonhematopoietic cells in various tissues was estimated by counting the proportion of CD45/ML-I-Y+ cells in 4 to 5 calves per tissue (including the exceptional case). Altogether, several tissues were analyzed from all 12 freemartins. Apart from FM10, the calves did not differ markedly in the relative frequency of CD45/ML-I-Y+ cells, which constituted less than 1% of all CD45/ML-I- cells in most tissues investigated (Fig. 2). There was no obvious correlation between the frequency of Y+ cells in blood and in nonhematopoietic tissues, or between different nonhematopoietic tissues in an animal. Donor-derived nonhematopoietic cells were markedly rare in the ectoderm-derived epidermis and CNS. Otherwise, no consistent differences between derivatives of different embryonic layers were seen. In 11 of 12 calves, highest numbers of CD45/ML-I-Y+ cells were detected in the connective tissue around ducts of mammary glands (0.94 ± 0.67%), liver (0.85 ± 0.41%, primarily not hepatocytes), and skeletal muscle (0.83 ± 0.26%). In cardiac and smooth muscle, renal epithelia, and epidermis the average frequency of CD45-Y+ cells was 0.1% or less (in epidermis the interdigitating Langerhans cells make the identification of CD45- cells difficult). In the brain, very few ML-I- donor-derived cells were found, although donor contribution to the microglia was in most cases substantial (we note that in addition to microglial cells, the lectin labels most endothelia). In the cerebral cortex and the underlying white matter, 0.0020 ± 0.0023% of ML-I- cells were Y+. In the hippocampus and the OB, two regions involved in the generation of new neurons in adult mammals , no ML-I-Y+ cells were detected in approximately 2 x 105 cells screened in double-stained sections of each tissue. No Y+ cells with clearly neuronal morphology were seen in about 720 successfully hybridized sections from freemartin CNS, representing approximately 28 x 106 total cells and including samples from cerebral cortex, cerebellum, brain stem, corpus callosum, hippocampus, OB, thalamus, ventricular walls, hypophysis, and spinal cord.
Figure 2. Proportions of bull-derived (Y+) cells in all nonhematopoietic (CD45/ML-I-) cells in 11 majority-type freemartins. Each tissue was analyzed from four majority-type calves (not the same calves for different tissues), with the exception of the mammary gland (three samples containing ducts available), and the regenerating tissue in implants (one sample per time point). Numbers were corrected with counts obtained from similar bull tissues as described in Materials and Methods. EP = epithelium, CT = connective tissue, MU = muscle tissue, NE = nervous tissue. For each tissue, individual samples are arranged in order of increasing animal age.
In FM10, the distribution of CD45/ML-I-Y+ cells differed dramatically from the rest of the calves. Higher numbers of CD45-Y+ cells were found in the epithelium of small intestine (19%, Fig. 1H), connective tissue of mammary gland (15%), liver (4.3%), epithelium of mammary gland (4.0%), skeletal muscle (2.3%), cardiac muscle (1.0%), and smooth muscle (0.5%), whereas in brain and in renal epithelia the frequency did not differ notably from the other calves. In the villi of the intestinal mucosa, strands of CD45-Y+ cells were seen (Fig. 1H). The hematopoietic system of FM10 differed from the majority of calves in that the donor contribution to B cells was clearly weaker than to the total leukocyte pool (not shown).
To investigate the role of donor-derived cells in tissue regeneration, we implanted cellulose viscose sponges in the subcutis of a 70-day-old freemartin and stained the invading granulation tissue for Y-chromosome and CD45. The regenerating tissue contained relatively high numbers of CD45-Y+ cells, with increasing numbers within the incubation period of 4 weeks (Figs. 1I, 2). At 4 weeks, the frequency of CD45-Y+ cells in the regenerating tissue was significantly higher than in most other nonhematopoietic tissues of the same animal (p = 0.12 for liver, p < 0.02 for any other tissue investigated). The frequencies of CD45/ML-I-Y+ cells in the tissues of the recipient did not differ markedly from those in the 10 other calves of the majority type. No clear Y+ cell clusters indicating local expansion were seen.
DISCUSSION
We thank M. Georges (University of Liège, Belgium) for the btDYZ clone, U. Pfüller (Witten/Herdecke University, Germany) for the biotinylated mistletoe lectin I, staff at the Saari unit (Dept. Clinical Veterinary Medicine, University of Helsinki) for providing blood samples of potential freemartins, K. Lahti, T. Pankasalo, H. Valtonen, and A. Koivunen for expert technical assistance, and L.C. Andersson and L.A. Lindberg for valuable comments about the work.
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