Target-antigen Detection and Localization of Human ...

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Annals of Clinical & Laboratory Science, vol. 45, no. 3, 2015

Target-antigen Detection and Localization of Human Amniotic-derived Cells after in Utero Transplantation in Rats Giovanni Pietro Burrai1, Elisabetta Antuofermo1, Serafina Farigu1,2, Anna Cargnoni2, Patrizia Bonassi2, Valeria Pasciu1, Maria Piera Demontis1, Ornella Parolini2, and Maria Vittoria Varoni1 1Dipartimento

di Medicina Veterinaria, Università degli Studi di Sassari, Sassari, and 2Centro di Ricerca E. Menni, Fondazione Poliambulanza-Istituto Ospedaliero, Brescia, Italy.

Abstract. Human amniotic-derived cells (hAMCs) have recently raised interest for their differentiation capability and immunomodulatory properties. To assess the feasibility of hAMCs therapeutic treatment during fetal development, we explored the localization of cells derived from the human amniotic membrane in rat organs after in utero transplantation. Rats were sacrificed at different time points and their organs were analyzed for the distribution of hAMCs by immunohistochemistry using an antibody against Human Cytoplasm and through detection of human DNA. Immunohistochemical and PCR analysis showed that most of the rat tissues presented human cells/DNA suggesting a widespread migration of hAMCs after transplantation. We developed an efficient target-antigen detection method based on an immunohistochemical technique that resulted to be highly specific and sensitive to identify the hAMCs into rat tissues. Key words: In utero transplantation, Immunohistochemistry, Human amniotic-derived cells, PCR, Placenta. Introduction Cells derived from the fetal tissues of human placenta and specifically from the amniotic membrane, have recently gained attention for their unique features which render them attractive for transplantation approaches and for regenerative therapies [6,22,24,25]. Apart from their relative ease of isolation and the lack of ethical concerns for human term placenta procurement, human amniotic derived cells (hAMCs) exhibit in vitro multilineage differentiation potential suggesting their utility in tissue regeneration approaches [22,23,26, 31].

[2,18,22,29]. These effects are most likely associated with the release of soluble factors, which in turn act in a paracrine manner to support survival and proliferation of host cells. Placenta-derived cells can secrete a number of factors with angiogenic [9], as well as anti-apoptotic and anti-oxidative action [13]. Therefore, due to these features, hAMCs have been generating and receiving increasing interest for their potential therapeutic use [23,24].

In addition, hAMCs demonstrate immunomodulatory properties, and in vitro studies show their capacity to inhibit lymphocyte proliferation [14] as well as to block differentiation of monocytes toward dendritic cells [15]. These features are the basis of their potential application in allogeneic transplantation settings and for their capacity to control in vivo inflammatory and fibrotic processes

Moreover, hAMCs could be promisingly applied in prenatal treatments such as in utero transplantation (IUT). Cell treatment during fetal life is mainly aimed to treat congenital disorders in the fetus in order to prevent organ damage due to the earlyonset genetic disorders. Furthermore, the delivery of cells into the fetus could induce immunotolerance allowing for the repetition/replication of the treatment postnatally. The fetal environment can also promote the proliferation and differentiation of transplanted cells and the fetal immature immune system can facilitate widespread engraftment [3,20].

Address correspondence to Dr. Giovanni Pietro Burrai, Dipartimento di Medicina Veterinaria, Università degli Studi di Sassari, Via Vienna 2, 07100, Sassari, Italy; phone: +39 079229438; fax: +39 079 229440; e mail: [email protected]

Even though few studies applied IUT as prenatal stem cell treatment using either hematopoietic [33] or mesenchymal cells from bone marrow [5,8],

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these reports suggest a promising use of stem cell IUT to treat different diseases such as immunodeficiencies [32], bonerelated diseases [7,8,10,22], metabolic diseases [27] and neural defects [28].

*GD: gestational day; †nt = not tested; Intensity of signals(PCR): +++ very bright; ++ bright; + clearly detectable; (+) detectable; - negative.

(+) (+) (+) (+) (+) (+) (+) Upper body +++ +++ +++ +++ +++ +++ Lower body +++ +++ +++ +++ +++ +++ +++ +++ Brain ++ ++ ++ ++ - + (+) Skin ++ ++ ++ ++ ++ ++ + + (+) - Skeletal muscle ++ ++ ++ - ++ ++ ++ + + + (+) (+) Lung ++ ++ ++ ++ ++ ++ ++ + + + Thymus ++ ++ ++ ++ ++ ++ + + Heart ++ ++ ++ ++ ++ ++ ++ + + + - Stomach ++ ++ ++ - ++ ++ ++ + + - Intestine ++ ++ ++ ++ ++ nt† ++ + - Spleen ++ ++ ++ ++ ++ ++ ++ + + (+) - Kidney ++ ++ ++ ++ ++ ++ ++ - Liver ++ ++ ++ ++ ++ + + Bone ++ ++ ++ nt† ++ ++ - + + + (+) Bone marrow ++ ++ ++ ++ ++ ++ ++ + + + + (+) -

11m 11l 10f 9h 9f 10c 10b 10a 9b 9a 8e 8d 8c 8b 7d 7c 7b 7a

Tissues/ID animal Fetuses at GD-19* Rats at birth

Table 1. PCR analysis of human DNA in rat tissues after human amniotic derived cells transplantation.

11a

11b

Rats at day 7 after birth

11f

9l

Rats at day 14 after birth

Immunohistochemical detection of human amniotic-derived cells

In addition to bone marrow-derived cells, other stem cell sources as placenta [3,4,16], adipose tissue [17] and amniotic fluid [28] have been transplanted in utero. Considering this potentiality, it is fundamental to develop animal models in order to investigate the potential efficacy of human cells as prenatal treatment for specific congenital pathologies and to study the migration, engraftment and functionality of these human cells after transplantation. Therefore, given the importance of the xenogeneic transplantation strategy, in this study we explored the migration and the localization of cells derived from the human amniotic membrane in host tissues/organs after in utero transplantation in rats. Unlike other authors who used labeled cells [4,16], we did not apply any in vivo cell tracking to identify hAMCs after transplantation, in order to minimally manipulate the cells and avoid any potential misinterpretation due to a possible transfer of stain to host cells [12]. Consequently we developed an immunohistochemical technique that we show to be highly specific and sensitive to identify human cells in rat tissues after transplantation. Materials and Methods Human amniotic cell (hAMC) isolation. Human term placentas were collected after obtaining informed written consent according to the guidelines set by the Ethics Committee for the Institution of Catholic Hospitals (CEIOC) and after their authorization to use placenta for experimental research (protocol number 16/2012), and were immediately processed.

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hAMCs were isolated from the epithelial and the mesenchymal layers of the human amniotic membrane as previously described [30]. Briefly, the amniotic membrane was manually separated from the chorionic membrane and washed in phosphate-buffered saline (SigmaAldrich, St. Louis, MO, USA) containing 100 U/ml penicillin and 100 μg/ml streptomycin (Lonza, Basel, Switzerland). Amniotic fragments (~3×3 cm) were enzymatically digested with 2.4 U/ml dispase (Becton Dickinson, Franklin Lakes, NJ, USA) for 9 min at 37°C, followed by a second digestion with 0.75 mg/ml collagenase (Roche, Mannheim, Germany) and 20 μg/ml DNAse (Roche, Mannheim, Germany) for 150 min at 37°C. The collagenase-undigested amniotic fragments were further incubated with 0.25% trypsin for 2 min at 37°C (Sigma-Aldrich, USA). All resulting cell suspensions were mixed, filtered through a 100 µm cell strainer and collected by centrifugation (300g, 10 min). These cells, obtained from the entire amniotic membrane, were cryopreserved in 10% DMSO supplemented with 90% FBS until use in cell transplantation procedures. Flow-cytometry analysis of cells revealed the following phenotype: CD73 (90±5%), CD166 (85±10%), CD90 (10±5%), CD13 (15±5%), CD45 and CD14 (1±0.5%). Immediately before transplantation, cryopreserved hAMCs were thawed and their viability was checked by Trypan blue exclusion. In all cases, at least 85% of the transplanted cells were viable. hAMCs were resuspended in Hanks' Balanced Salt Solution (HBSS) with a final concentration of 2x105 cells in 10 µl of HBSS. Animals and experimental groups. All animal experiments were performed according with the Directive 2010/63/EU for animal experiments with the approval of the Italian Ministry of Health (protocol number 633/10). Five pregnant Sprague Dawley rats (Charles River, Calco, Lecco, Italy) were housed at room temperature (24±1°C), with constant humidity (60%±5) and a light/dark cycle of 12 h. Animals had free access to tap water and rat chow (standard diet; Mucedola, Milan, Italy). On gestation day (GD) 18, rats were anesthetized by im injection of zoletil (25 mg/Kg) plus xilazine (1 mg/Kg) and uterine horns were exposed through a midline laparotomy incision. Using a 30 gauge needle (Hamilton, Reno, Nevada), each fetus was injected through the uterine wall with 2x105 of hAMCs in 10 µl of HBSS. In the first rat, fetuses of one horn were not injected and used as negative controls. Then the uterus was gently replaced and abdominal tissues sutured.

Twenty-four hours after transplantation (at GD-19), two pregnant rats underwent laparotomy under deep anesthesia. The fetuses were extracted and the number of live fetuses in each uterine horn was recorded. Half of the fetuses, after sacrifice, were cryopreserved at -80°C for PCR assay and the other half was formalin fixed for evaluation by histology and immunohistochemistry. Rats born from the other 3 rat dams were sacrificed at different time points after birth (0, 7 and 14 days corresponding to 4, 11 and 18 days post in utero transplantation) and complete necropsies were performed. Rat tissues (skin, skeletal muscle, bone and bone marrow) and organs (brain, lung, thymus, heart, stomach, intestine, spleen, kidneys and liver) were used for PCR analysis and immunohistochemistry. Detection of human DNA in rat tissues. The presence of human cells was assessed in rat tissues collected from transplanted animals at GD-19, at birth, and after 7 and 14 days from birth by PCR analysis of human mitochondrial cytochrome B gene [21]. Total DNA (50–100 ng) was extracted from rat tissues using a Bio robot EZ1 (Qiagen, Hilden, Germany) and the EZ1Tissue Kit (Qiagen) according to the manufacturer’s instructions, and was then amplified in 50 μl reactions containing dNTPs (200 μmol), GoTaq DNA polymerase reagents (Promega, Madison, WI, USA), and the following primers (25 pmol) specific for the human sequence of the cytochrome B mitochondrial gene: 5′-CCCATACATTGGGACAGACC-3′ (forward) and 5′-GACGGATCGGAGAATTGTGT-3′ (reverse) [21]. All PCR reactions included an initial step at 95°C for 10 min. This was followed by 40 cycles (94°C for 30 s, 58°C for 30 s, 72°C for 1 min) for DNA amplification of human mitochondrial cytochrome B. PCR products were analyzed by 1.8% agarose gel electrophoresis and ethidium bromide staining, followed by Southern blotting. For Southern blot analysis, PCR products were transferred to nylon membranes (Hybond N+ Amersham Biosciences, Little Chalfont, UK) and hybridized to specific probes labeled with horseradish peroxidase enzyme (ECL Gold, Amersham Biosciences). Chemiluminescent signals were detected with a Gel Doc 2000 system (BioRad, Hercules, CA). Histology. Eight fetuses at GD-19 were sacrificed, half sectioned and processed for histology. Seven rats at birth, as well as 4 and 3 rats were sacrificed at 7 and 14 days after birth, respectively. Tissues from rats were fixed in 10% neutral buffered formalin, paraffin-embedded, and four serial sections (3µm-thick) were

Immunohistochemical detection of human amniotic-derived cells Table 2. Human amniotic derived cell distribution in fetuses and rats detected by immunohistochemistry (STEM 121) after different times from transplantation. Organs Fetuses Rats positive Rats positive at at birth positive GD-19* No. (%) at day 7 No. (%) after birth No. (%) Brain Lung Pleura Heart Omentum Liver

1/8 (12.5%) 5/8 (62.5%) 1/7 (14.3%) 5/8 (62.5%) 1/7 (14.3%) 1/8 (12.5%) 3/8 (37.5%) 1/7 (14.3%) 3/8 (37.5%)

*GD: gestational day cut at 50 micrometer intervals. Slides were stained with hematoxylin and eosin (H&E) and Masson's trichrome to evaluate tissue morphology. Photomicrographs were acquired with a Nikon Digital Sight DS-U1 camera mounted on a Nikon 80-i microscope. Immunohistochemistry. Sections from formalin-fixed, paraffin-embedded tissues were mounted on positively charged Superfrost slides (Fisher Scientific). Slides were immersed for 20 min in a 98°C, preheated solution (WCAP, citrate pH 6, BiOptica, Milan, Italy) that simultaneously allows dewaxing, rehydration and antigen unmasking. Briefly, slides were mounted in a humidity chamber (Shandon, Runcorn, UK) and tissues were then blocked for endogenous peroxidase with a 15 min incubation in Dako REAL Peroxidase-Blocking Solution (S2023, Dako, Glostrup, Denmark), and for non-specific binding with 2.5% normal horse serum (ImmPRESS reagent kit,Vector Labs, Burlingame, CA, USA) for 30 minutes at room temperature. Then, sections were incubated overnight at 4°C with a mouse monoclonal Human Cytoplasmic Marker (STEM 121 StemCells, Inc. Cambridge, UK) and incubated for 20 min at room temperature with an anti-mouse rat absorbed secondary antibody (MP-7422, ImmPRESS reagent kit, Vector Laboratories, Burlingame, CA, USA). 3,3'-Diaminobenzidine (DAB) (ImmPACT DAB, Vector Laboratories, Burlingame, CA, USA) was used as the chromogen. All washing steps were performed three times with TBS-0.1% Tween 20 (BiOptica, Milano, Italy). Tissues were counterstained with haematoxylin, dehydrated and mounted with Eukitt® Mounting Medium (BiOptica, Milan, Italy). A human amnion

1/4 (25%)

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sample served as positive control, while negative control samples were obtained from untreated rats. Cytoplasmic immunostaining and localization of human amniotic-derived cells by STEM 121 in organs of rats were evaluated counting the total immunolabeled cells in four consecutive sections. Samples were considered positive if at least one immunostained cell was detected in the four analyzed sections. The slides were scored independently by two pathologists (GPB and EA) and a consensus score was obtained using a multi-head microscope.

Results

Animal survival. Similarly to previous reports [4], we observed a fetal death rate of 18% at GD-19 (when fetuses were extracted from anesthetized pregnant rats after 24 hours from transplantation) and 47% at birth. Presence of human DNA in rat tissues after hAMC transplantation. As reported in Table 1, twenty-two animals treated in utero with hAMCs were evaluated for human DNA distribution in various rat tissues by PCR analysis using the human specific mitochondrial cytochrome B gene probe. The analyses were performed in all organs/ tissues listed in Table 1, collected from 8 fetuses at GD-19, from 7 rats at birth, from 4 rats at day 7 after birth, and from 3 rats at day 14 after birth. All rat fetuses (8/8) examined at GD-19 showed high human DNA. At birth, i.e. 4 days after transplantation, human DNA was detected in various tissues and organs of all analyzed rats (7/7), indicating that transplanted cells were capable of widespread migration. In particular, a marked positivity for human DNA was found in the lung, heart, intestine, spleen, kidney and bone marrow of all animals analyzed; in the thymus, skin, stomach and skeletal muscle of 6/7 animals; in the bone of 5/6 animals; in the liver of 5/7 animals, and in the brain of 4/7 animals examined. Seven days after birth, all rats continued to show clearly detectable distribution of human DNA in the bone marrow, 3/4 animals in the lung, bone, heart

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Figure 1. A. Lung; B. Pleura: human amniotic derived cells detected by immunohistochemistry with STEM 121 in a fetus at gestation day 19. Several cells showed a strong cytoplasmic staining. Immunoperoxidase-DAB. Scale bar = 50 μm.

Figure 2. A. Pleura: human amniotic derived cells detected by immunohistochemistry with STEM 121 in a rat sacrificed at birth. Immunoperoxidase-DAB. Scale bar = 50 μm. B. Proliferation of fibrous connective tissue (arrow) in lung parenchyma and pleura. Masson's trichrome stain. Scale bar = 50 μm.

Figure 3. A and B. Omentum: few human amniotic derived cells were detected by immunohistochemistry with STEM 121 in a rat sacrificed at day 7 after birth. Mild lymphocytic infiltrate is also present. Immunoperoxidase-DAB. Scale bar =100 μm (A) and =10 μm (B).

Immunohistochemical detection of human amniotic-derived cells

and skeletal muscle; 2/4 in the thymus, skin, stomach, spleen and liver; 1/4 in the intestine and brain, while no animal presented human DNA in the kidney. Fourteen days after birth, human DNA was detected with a lower intensity and in a reduced number of host tissues. However, all of the animals analyzed (3/3) still showed human detectable DNA. In particular, human DNA was detected in the skeletal muscle of all rats, in the skin, bone, brain and bone marrow of 2/3 rats examined, and in the thymus, stomach and spleen of 1 animal. Histological and immunohistochemical results. Human amniotic-derived cells were strongly detectable in rats using the mouse monoclonal Human Cytoplasmic Marker (STEM 121). hAMCs were observed in all fetuses at GD-19 (8/8), in 43% (3/7) of rats examined at birth, in 25% (1/4) of rats at day 7 after birth, and in 0% (0/3) of rats at day 14 after birth. Most fetal tissues had detectable human amnion-derived cells at GD-19, whereas in rats at birth and at day 7 after birth only a few organs showed immunostained detectable cells (Table 2). Both in rats sacrificed at GD19 (Figures 1A and B) and rats sacrificed at birth (Figures 2A and B) the higher amount of human cells was detected in the lungs (31.2±17.4 cells, mean ±SEM) and in the pleura (15.4±10.3 cells, mean ±SEM). The omentum presented the most prolonged presence of human cells being positive also in animals sacrificed 7 days after birth (see Table 2). Furthermore, in rats sacrificed at birth, hAMCs were also associated to a mild lymphocytic infiltrate and scant connective tissue deposition (Figure 2B), evident also in the omentum of rats sacrificed at day 7 from birth (Figures 3A and B).

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fetuses with congenital disorders at an early-stage of the pathology, during the prenatal life [3,20]. Considering this potentiality, it is fundamental to develop xenograft animal models in order to investigate the potential of the prenatal treatment performed with human cells on specific congenital pathologies and one relevant question to address is cell homing after human cell transplantation. Thus, in this study, we sought to investigate the migration and the localization of cells derived from the human amniotic membrane, by PCR and immunohistochemistry analysis, at different time points after xenogeneic in utero transplantation in rats. Several cell tracking methods have been applied to detect hAMCs after transplantation. Most of those methods typically consisted of transplantation of labeled cells [4,16]. However, as recently reviewed [12], there are several points to be taken into consideration such as the shortcomings or the lack of specificity of the labeling dyes. In addition, most of these labeling dyes are endogenously expressed in the host tissue and often complicate data interpretation [12].

Discussion

In our study, we used a monoclonal antibody that is raised against a cytoplasmic protein of human cells and does not cross-react with tissue from mouse or rat. This antibody allowed us to detect hAMCs at different time points after transplantation in different tissues/organs of transplanted animals. Immunohistochemical and PCR analysis showed that most of the rat tissues presented human cells and DNA at different time points indicating that hAMCs can migrate and colonize specific organs, whether this could result in their capacity to acquire the specific characteristics/phenotypes of the various tissues, or in their homing to exert the well described paracrine effect due to their secreted factors remains to be determined [29].

Human amniotic membrane-derived cells are receiving renewed interest in the field of regenerative medicine and cell transplantation mainly for their immunomodulatory and multilineage differentiation properties [22,30]. One interesting application of these cells, even though not yet fully investigated, is the in utero transplantation aimed to treat

Through the immunohistochemical analysis, hAMCs were found in all fetuses examined 24 h after cell transplantation, in 43% of rats examined at birth, in 25% of rats sacrificed at day 7 after birth, and hAMCs were not found in rats sacrificed at day 14 after birth. PCR analysis showed positivity for human DNA sequence in most of the

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examined tissues, as previously reported [3,4]. The presence of human cells determined by immunohistochemistry decreased after 7 days from birth and no evidence of transplanted cells was found in rats sacrificed after 14 days from birth. PCR analysis showed a similar pattern with a decrease of signal intensity with the progression of the time from cell transplantation, however, due to the higher sensitivity of the PCR [30], positive signals were still observed up to the last time point. Even though PCR, as expected, was more sensitive and indeed revealed higher positivity than IHC, the localization and quantification of cells was achieved exclusively using IHC.

Due to the limitation of the number of the animals studied, we cannot make conclusions on the engraftment potential of hAMCs, even though from these preliminary data we could confirm what observed in other models [3,4,16]. In this study we provide an efficient and reliable target-antigen detection method that could be used to provide new insights into the potential of human amniotic cells in transplant settings. This study adds to continuing efforts by different investigators to explore the potential of in utero cellular transplantation, and warrants further investigation of using fetal membrane-derived cells for prenatal cell therapies.

These results are in agreement with previously reported data [4], who observed that the number of human placenta-derived cells detectable in the host rat tissue after transplantation showed a downward trend. Thus, the two methodologies applied in this study to identify the transplanted hAMCs showed a general agreement in the detection of host cells in rat tissues.

We thank Regione Autonoma della Sardegna (L.R. 7/2007 Bando 2008) for its support for this research.

In addition, most hAMCs detectable by immunohistochemistry appear as a single cell or, more often, as a cluster suggesting a more extensive investigation of whether this may be an indication of transplanted cell proliferation, as recently suggested by Caruso and colleagues [3]. Although the distribution pattern and number of cells in the individual fetuses varied, hAMCs were detectable mostly in the lungs and pleura suggesting that these cells are able to migrate to these sites, as previously reported [1,19]. This capacity has been exploited by several authors to efficiently treat lung injuries such as pulmonary fibrosis in a rat bleomycin-induced model [2,18]. However, differently to previous reports, in rats sacrificed at birth, after 24h from cell transplantation, hAMCs were also associated to a mild lymphocytic infiltrate and connective tissue deposition, evident also in the omentum of rats sacrificed 7 days after birth. Larger, carefully designed studies are needed to further investigate this result.

Acknowledgment

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