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Hematopoietic stem cell engraftment by early-stage in utero transplantation in a mouse model
Experimental and Molecular Pathology 87 (2009) 173–177
Contents lists available at ScienceDirect
Experimental and Molecular Pathology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x m p
Hematopoietic stem cell engraftment by early-stage in utero transplantation in a mouse model Xin Chen a,c, Xiu-li Gong a,c, Makoto Katsumata a,d, Yi-tao Zeng a,c, Shu-zhen Huang a,c,⁎, Fanyi Zeng a,b,c,⁎ a
Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Institute of Medical Science, Shanghai Jiao Tong University School of Medicine, Shanghai, China Key Laboratory of Embryo Molecular Biology, Ministry of Health of China, Shanghai, China d Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA b c
a r t i c l e
i n f o
Article history: Received 9 July 2009 Available online 7 August 2009 Keywords: In utero transplantation Hematopoietic stem cell Chimera establishment Allogeneic transplantation Fluorescence in situ hybridization
a b s t r a c t A novel intrauterine transplantation (IUT) approach was developed to improve the efficiency of engraftment of hematopoietic stem cells (HSCs). HSCs with a green fluorescent protein (GFP) reporter gene were transplanted in utero on days 12.5, 13.5 and 14.5 post coitum (p.c.). The degree of chimerism of donor cells in recipient newborn mice was examined using fluorescent microscopy, polymerase chain reaction (PCR), fluorescence-activated cell sorting (FACS), and fluorescence in situ hybridization (FISH) analyses. Microscopic examination revealed the presence of green fluorescent signal in the peripheral blood of the chimeric mice. The highest survival rate (47%) as well as the highest chimerism rate (73%) were achieved by our new approach in the newborn mice that were subjected to in utero transplantation (IUT) on day 12.5 p.c. (E12.5) compared to the conventional IUT method. FACS analysis indicated that 1.55 ± 1.10% of peripheral blood cells from the newborn mice were GFP-positive donor cells. FISH showed that cells containing the donor-specific GFP sequence were present in the bone marrow (BM) of the chimeric mice. Thus, the efficiency of chimera production with this new method of IUT was significantly improved over the existing IUT techniques and instruments. © 2009 Elsevier Inc. All rights reserved.
Introduction As a result of rapid advances in molecular and diagnostic medicine, a growing list of hereditary defects can be diagnosed before birth (Zeng and Huang, 1985; Modell et al., 1998; Flake and Zanjani, 1997). Prenatal diagnosis offers the opportunity for early management of those diseases. IUT of stem cells has been studied as one of the approaches for treating a range of genetic birth defects, including hemoglobinopathies, immunological defects and inborn errors of metabolism (Flake et al., 1996; Muench and Bárcena, 2004; Muench, 2005). However, prenatal treatment still faces many challenges due to poor understanding of stem cell behavior in vivo (Brännström and Wranning, 2007). Therefore, establishment of an effective animal
Abbreviations: IUT, in utero transplantation; HSC, hematopoietic stem cell; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; PCR, polymerase chain reaction; FACS, fluorescence-activated cell sorting; FISH, fluorescence in situ hybridization; BM, bone marrow; PBS, phosphate buffered saline; MNC, mononuclear cell; MACS, magnetic-activated cell sorting; PB, peripheral blood; RBC, red blood cell. ⁎ Corresponding authors. S. Huang is to be contacted at Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University, 24/1400 West Beijing Road, Shanghai 200040, China. Fax: +86 21 6247 5476. F. Zeng, Institute of Medical Science, Shanghai Jiao Tong University School of Medicine, 227 South Chongqing Road, Shanghai 200025, China. Fax: +86 21 6247 5476. E-mail addresses: [email protected] (S. Huang), [email protected] (F. Zeng). 0014-4800/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2009.07.009
model of IUT for the studies of homing, differentiation and proliferation of the donor stem cells would provide great insights into the prenatal treatment of many genetic diseases. In the past decade, IUT has been utilized in primates, sheep, goats, rats, and mice (Harrison et al., 1989; Zanjani et al., 1994; Zeng et al., 2005; Rice et al., 1994; Pixley et al., 1994). We have successfully established an experimental model of transplanting human HSCs into fetal goats using B-scan ultrasonography (Zeng et al., 2006). In the present study, we attempted to establish an IUT model in mice, which are suitable for studies of prenatal treatment because of their small size, short breeding cycle and defined genetic background. However, performing IUT in mice is much more challenging because the fetus is small and requires precise and skillful surgical techniques. As a result, the survival and chimeric rates are often very low. The purpose of this investigation is to establish an IUT procedure that can improve the success rate and achieve high levels of chimerism. To this end, we developed a new intrauterine transplantation instrument and modified existing surgical procedures. We assessed the improvement of the survival rate and degree of chimerism of engraftments of murine BM-derived HSCs by performing various molecular analyses. This new IUT methodology allows the efficient generation of chimeric mice that will provide a useful model for the studies of stem cell biology in vivo.
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Materials and methods Preparation of donor cells Donor cells were prepared from transgenic mice (4–8 months of age) obtained from the Shanghai Institute of Medical Genetics. The mice had an EGFP gene controlled by the β-globin promoter/ enhancer in which the EGFP (i.e., HS23-eGFP vector) is expressed in an erythroid-specific manner (Jia et al., 2003). Donor bone marrow (BM) was harvested sterilely by flushing the tibias and femurs with phosphate buffered saline (PBS) using a 26-gauge needle. A single cell suspension was prepared by 3 gentle passes through the needle. Cell suspensions were then filtered through a 70-μm nylon mesh and layered over Ficoll (1.077 g/ml; Axis-Shield PoC AS, Oslo, Norway). After centrifugation at 600 g for 20 min, the mononuclear cell (MNC) layer was removed and washed twice with PBS. Sca-1+ (mouse hematopoietic stem cell antigen) cells were enriched from MNCs by magnetic-activated cell sorting (MACS) with a Sca-1 progenitor cell isolation kit (Miltenyi Biotec, Bergish Gladbach, Germany) according to the manufacturer's instructions. The cells were counted prior to transplantation, and greater than 95% viability was confirmed by a trypan blue exclusion assay. Mice Mice of the Kun-Ming Bai strain (8–10 weeks old) were obtained from the animal facility of Shanghai Institute of Medical Genetics, Shanghai Children's Hospital; and their utilization was approved by the Review Board of Shanghai Children's Hospital. Fetuses of timed pregnant mice were transplanted on days 12.5 (E12.5), 13.5 (E13.5) and 14.5 (E14.5) p.c. (term gestation 21 days). Development of a new instrument for in utero transplantation A new instrument was designed as follows: a sterile plastic tube (Westingarea Corporation, China) was cut to 7–8 cm long. Its standard scalp interface end was connected to a 10 μl capacity adjustable pipettor; while the other end was connected to a 33-gauge injection needle (Popper & Sons, Germany). The injection volume was controlled via the pipettor (Supplementary Figs. 1B and C). Procedures for in utero transplantation
Fig. 1. In utero transplantation of murine Sca-1+ cells into fetal peritoneal cavity. The fetuses are injected using the novel injection instrument while the subject fetus is immobilized with a sterile cotton swab (A). The arrow indicates the injection site (B).
Identification of donor cell engraftment
Nested-PCR analysis DNA was extracted from blood cells of recipient mice. The GFP sequence was detected by a nested-PCR procedure using sequence specific primers (GFP primers: S1, 5′-TGACCTACGGCGTGCAGTGCTT3′; A1, 5′-TCGTCCATGCCGAGAGTGATCC-3′; S2, 5′-CACATGAAGCAGCACGACTTCT; and A2, 5′-AACTCCAGCAGGACCATGTGAT-3′). The amplification of sequence was as follows. In the first PCR, 15 μl of PCR mixture with the primers S1 and A1 was used. After the initial denaturing step at 94 °C for 5 min, 16 cycles of amplification were performed, including 45 s at 94 °C, 45 s at 58 °C, 1 min at 72 °C, and a final extension at 72 °C for 10 min. In the second PCR amplification, 25 μl of a mixture containing the primers S2 and A2 and 5 μL of the first PCR products was used. PCR conditions were similar to those of the first PCR except for the annealing step for 30 s. In total, 28 cycles were performed. As a control, β-actin primers were used (S, 5′CGCTCGTTGCCAATAGTGAT-3′; A, 5′-CCACAGGCATTGTGATGG-3′) with the annealing temperature at 60 °C for 28 cycles. The PCR products were separated in a 2.0% agarose gel and visualized by ethidium bromide staining.
Fluorescent cells analysis Blood samples (4–5 μl) were collected in heparinized microhematocrit tubes. Peripheral blood smears were prepared, air dried and fixed with 95% ethanol. GFP-red blood cells (RBCs) were identified using a Leica DM RXA2 fluorescent microscope.
Fluorescence-activated cell sorting (FACS) analysis FACS analysis was performed after birth at set intervals using approximately 10 μl of peripheral blood (PB) collected in heparinized capillary tubes via retro-orbital vein puncture of mice. The blood was then analyzed with flow cytometry to determine the presence of donor
Murine bone marrow-derived Sca-1+ cells were injected directly into the fetal peritoneal cavities. On E12.5, E13.5 and E14.5, pregnant mice were anesthetized with 1% pentobarbital sodium intraperitoneally (i.p.) at 8 ml/kg pre-pregnancy body weight. Using sterile technique, a midline laparotomy was made and the uterine horns were visualized. Each fetus was administered with 5 × 104 Sca-1+ cells i.p. in 3 μl of PBS through the intact uterine wall using the improved transplantation needle by a two-person operation, one responsible for the injection, and the other one for pipette control (Figs. 1A and B). Control animals received 3 μl PBS instead of the donor cells. The uterus was carefully placed back into the maternal peritoneal cavity, followed by abdominal closure. The mothers were kept warm until they recovered from anesthesia. The pups were carried to term, delivered normally, and subsequently weaned at 3 weeks of age.
X. Chen et al. / Experimental and Molecular Pathology 87 (2009) 173–177 Table 1 Survival and chimerism rates after IUT of murine Sca-1+ cells using the conventional vs. novel transplantation instruments. Group
No. of fetuses injected
No. of live-born pups
Survival rate (%)
Chimerism rate (%)a
Conventional Novel
81 73
28 44
34 60⁎
29 53⁎
The fetuses at day 14.5 of gestation were transplanted intraperitoneally with 5 × 10 murine BM-derived Sca-1+ cells. a Status of chimerism was confirmed by molecular biological methods. ⁎ p b 0.05 between the two groups.
4
GFP-RBCs. One hundred thousand cells were used per measurement, and duplicate measurements were performed for each sample. Fluorescence in situ hybridization (FISH) analysis The mouse BM was used for FISH analysis according to the methods described in our previous study (Zeng et al., 2005). To detect the donor GFP cell chimera, the HS23/EGFP vector was labeled with DIG-Nick Translation Mix (Roche, Germany) according to the manufacturer's protocol. FISH signals were examined with a Leica DM RXA2 fluorescent microscope. Images were captured with the photography system associated with the microscope.
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survival birth rate was significantly higher (60% vs. 34%, p b 0.05). Nested-PCR and FACS analyses were performed to verify the existence of donor cells in the peripheral circulation of the recipient mice. The rate of chimerism in the experimental group was significantly higher than that of the conventional group (53% vs. 29%, p b 0.05; Table 1). IUT of murine BM-derived Sca-1+ cells into the peritoneal cavities of fetal mouse recipients at different developmental stages We first examined the effect of IUT on stem cells as well as our technical consistency with the novel IUT instrument. Each recipient fetus received approximately 5 × 104 Sca-1+ cells in 3 μl of PBS at E12.5, E13.5 or E14.5 stage. A comparable number of control animals received the same volume of PBS. The survival rate of the Sca-1+ cell injected group at E12.5 was 47% (32/69), at E13.5 was 44% (27/62) and at E14.5 was 60% (44/73), while that of the control group was 49% (32/65), 49% (36/74) and 58% (39/68) at E12.5, E13.5 and E14.5, respectively. These survival rates were not significantly different between the two groups in each developmental stage (p N 0.05; Fig. 2A). The status of the engraftment of donor cells was examined by nested-PCR and FACS analyses. The results showed that the chimerism rate when we transplanted at E12.5 was 73%, which was significantly higher than that of E14.5 (53%; p b 0.05; Fig. 2B). Fluorescent microscopy analysis of peripheral GFP cells in chimeric mice
Statistical analysis All the data were presented by plotting the mean value and standard deviation of each group. Statistical comparisons between the groups were performed by chi-square test or two-tailed Student's t-test assuming unequal variances, and a p-value of b0.05 was considered significant.
The presence of donor GFP-RBCs in peripheral blood of the transplanted mice was confirmed by fluorescent microscopy. The results demonstrated that GFP-RBCs were present in the peripheral blood of transplanted mice but not in control mice. This illustrated that the hematopoietic stem cells of donor origin could differentiate into mature erythrocytes expressing GFP in transplanted mice (Fig. 3).
Results Nested-PCR analysis of donor genomic DNAs Construction of the transplantation instrument for IUT with murine BM-derived Sca-1+ cells The conventional instrument for IUT consists of a glass capillary (Supplementary Fig. 1A). In our injection instrument, the glass capillary was substituted with a 33-gauge injection needle to minimize the damage to the fetus. The amount of the HSCs to be injected was controlled by a pipette (Supplementary Figs. 1B and C). Both instruments were used for IUT on E14.5 fetuses. For the experimental group, we transplanted Sca-1+ cells into 73 fetuses in utero using the novel instrument, and 44 pups were born at term. In contrast to the group with the conventional injection method, the
Nested-PCR was carried out on newborn mice transplanted at 12.5 days of gestation to verify the existence or expansion of grafted donor cells in the peripheral circulation. Nineteen out of the twentysix transplanted mice showed donor GFP chimerism in their peripheral blood while the control group was negative (Fig. 4). FACS analysis of chimerism rate in recipients In order to confirm the stability of the donor cell engraftment, blood samples from the transplanted mice were examined by a series of flow cytometry analysis at 1, 4, 8 and 12 months of age. The average
Fig. 2. Comparison of survival and chimerism rates between murine Sca-1+ cell injected and control injected groups after IUT at different developmental stages. The newborn survival rates are not significantly different between the two groups (p N 0.05, n N 60) at each stage (A). The highest chimerism (73%) is achieved at E12.5, and this rate was significantly higher than that of E14.5 (p b 0.05, n N 60) (B).
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Fig. 3. Fluorescent microscope analysis of donor GFP cells in peripheral blood (PB) of transplanted mice (40×). TM = GFP transgenic mouse, SCT = transplanted mouse, NM = untransplanted mouse. Fluorescence only (A). Fluorescence + light (B). GFP-positive cells are identified in the PB of the transplanted mice, but not in the untransplanted mice.
proportion of donor GFP cells was 1.55% [mean ± SD = (1.55 ± 1.1)%] (Fig. 5). All the cells in peripheral blood from the control group of untransplanted mice were GFP negative. FISH analysis of cells with donor-specific GFP signals To further confirm the presence of donor cells, we performed interphase FISH experiments. A DNA probe specifically detected donor HS23/EGFP vector signals in BM cells of the HSC transplanted mice, but not in untransplanted mice (Fig. 6). Discussion Some cattle twins are dizygotic yet share placental circulation, a condition known as freemartinism. Persistent chimerism was documented in freemartin cattle, indicating that a significant exchange of cells occurred during development, which can result in the acquisition of donor-specific immune tolerance (Owen, 1945). The goal of fetal IUT is to mimic the effects of placental cross-circulation by directly transplanting cells into a fetus, thereby generating chimerism. In the IUT procedure, donor cells are transplanted by intraperitoneal (i.p.) injection. However, in the mouse experimental system, the IUT operation is rather difficult because the size of the fetuses is very small and there are multiple fetuses per pregnancy (regularly 6–10 fetuses). In the IUT procedure, a small glass needle is traditionally employed. The glass needles are intrinsically fragile, and it is cumbersome to adjust the injection volume using a syringe attached
Fig. 4. Detection of donor-specific GFP sequence in the transplanted mice by nestedPCR. Lane M: 100 bp DNA marker; lanes 1, 4–11: nine individually transplanted mice; lane 2: blank; lane 3: GFP transgenic mouse; lane 12: untransplanted mouse. Beta-actin DNA was amplified as an internal control.
at the other end of the needle (Wang et al., 2005). As a result, the process is often unnecessarily prolonged, resulting in increased risk of fetal death and later abortion. To circumvent these problems, we systematically changed the design of the injection instrument and operational scheme. We utilized the smallest sterile disposable injection needle (33-gauge) to minimize the damage to the fetuses. Then, we connected a 10 μl capacity adjustable pipettor to the injection needle so that the total volume to be injected could be accurately controlled by adjusting the pipettor. We also shortened the total time necessary for a series of injections for all fetuses to less than 10 min by the two-person operation mentioned in the Materials and methods section. The efficiency of the IUT appears to be significantly improved with these modifications. IUT is a traumatic operation compared with some other surgeries, and a couple of important technical details should be noted for the success of this procedure. Close monitoring of the anesthesia state is essential in addition to the choice of the anesthetics and doses. In the pregnant mice, the fetuses account for a large part of the weight. The initial dose of anesthetic should be calculated based on the prepregnancy weight of the recipient mice, and depth of the anesthesia should also be monitored periodically throughout the procedure by pinching toes or observing the breathing rate. Additional anesthetic
Fig. 5. FACS analysis of donor cell engraftment in recipient mice at 1, 4, 8 and 12 months of age. Nineteen different dots represent proportion of donor GFP cells in nineteen individually transplanted mice. No GFP cells were detectable in untransplanted mice.
X. Chen et al. / Experimental and Molecular Pathology 87 (2009) 173–177
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Fig. 6. Fluorescence in situ hybridization (FISH) of the BM cells of representative mice. TM = GFP transgenic mouse. Each of the BM cells at interphase shows one hybridization signal. SCT = transplanted mouse. One of the BM cells in the field shows hybridization signal. NG = untransplanted mouse. None of the BM cells examined shows hybridization signal.
needs to be administered as necessary. Routine preoperative care, e.g. administration of analgesia and aseptic surgical environment, is also important. The other factor is the intra- or postoperative care. Close monitoring and maintenance of normal body temperature of the recipient mouse during and after surgery are important to prevent abortion. Providing a clean and quiet housing environment, highenergy diet with supplements and administration of an analgesic are also important for successful pregnancy. Our current investigation clearly showed that the developmental stage at which IUT is performed on the fetal recipients is the most important factor for a successful engraftment of HSCs. Pregnancy is divided into three stages; namely, early, medium and late periods. For most large animals, IUT is performed at the early period when the immune system of the fetuses is immature and the donor stem cells are easier to engraft in the host. However, in the mouse, IUT has been performed at the mid period (typically at E14.5) because the basic form of the fetuses at the early stage (before E7) is too small to distinguish (Hayashi et al., 2002; Frattini et al., 2005). In our current experiment, we were able to perform IUT at E12.5 for the first time. When the efficiency of the IUT in terms of survival rate and the degree of chimerism was compared among fetuses subjected to IUT at E12.5, E13.5 and E14.5, it is important to note that while the survival rates did not decrease at all for fetuses with IUT at E12.5, the degree of chimerism at this stage was the highest. Fetal immunologic tolerance might have played an important role for this improvement in relation to the degree of fetal thymic development (Peranteau and Flake, 2006). Thus, by not compromising the survival of the embryos at this earlier developmental stage, we significantly improved the IUT efficiency by producing a higher degree of chimerism. We successfully verified this by fluorescent microscopy, nested-PCR, and FACS, as well as FISH analyses. It should be noted that our technological advancement in designing the novel injection instrument and scheme contributed to the creation of mice with higher levels of allogeneic chimerism following IUT. In conclusion, we successfully modified and improved the IUT instruments and procedures for efficient allogeneic transplantation with a high success rate and degree of chimerism in order to study the biological properties of hematopoietic stem cells in vivo in a mouse model. Intrauterine stem cell transplantation provides a great potential advantage in prenatal treatment. In the near future, with the in-depth study of stem cell biology, IUT could be routinely applied in clinical treatments of many genetic defects. Acknowledgments This work was supported by grants from the National Key Scientific Research Program of China (2007CB947800 to FZ) and National Basic Research Program of China (2007CB511904 to SH). We especially thank Professor Hao Shen for his helpful discussion for this study. We also thank Mr. Yin Cheng for his excellent technical support.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexmp.2009.07.009. References Brännström, M., Wranning, C.A., 2007. Uterus transplantation: where do we stand today and where should we go? Expert Opin. Biol. Ther. 7, 427–429. Flake, A.W., Roncarolo, M.G., Puck, J.M., Almeida-Porada, G., Evans, M.I., Johnson, M.P., Abella, E.M., Harrison, D.D., Zanjani, E.D, 1996. Treatment of X-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow. N. Engl. J. Med. 335, 1806–1810. Flake, A.W., Zanjani, E.D., 1997. In utero hematopoietic stem cell transplantation: a status report. J. Am. Med. Assoc. 278, 932–937. Frattini, A., Blair, H.C., Sacco, M.G., Cerisoli, F., Faggioli, F., Catò, E.M., Pangrazio, A., Musio, A., Rucci, F., Sobacchi, C., Sharrow, A.C., Kalla, S.E., Bruzzone, M.G., Colombo, R., Magli, M.C., Vezzoni, P., Villa, A., 2005. Rescue of ATPa3-deficient murine malignant osteopetrosis by hematopoietic stem cell transplantation in utero. Proc. Natl. Acad. Sci. U. S. A. 102, 14629–14634. Harrison, M.R., Slotnick, R.N., Crombleholme, T.M., Golbus, M.S., Tarantal, A.F., Zanjani, E.D., 1989. In-utero transplantation of fetal liver haemopoietic stem cells in monkeys. Lancet 2, 1425–1427. Hayashi, S., Peranteau, W.H., Shaaban, A.F., Flake, A.W., 2002. Complete allogeneic hematopoietic chimerism achieved by a combined strategy of in utero hematopoietic stem cell transplantation and postnatal donor lymphocyte infusion. Blood 100, 804–812. Jia, C.P., Huang, S.Z., Yan, J.B., Xiao, Y.P., Ren, Z.R., Zeng, Y.T., 2003. Effects of human locus control region elements HS2 and HS3 on human β-globin gene expression in transgenic mouse. Blood Cells. Mol. Dis. 31, 360–369. Modell, M., Wonke, B., Anionwu, E., Khan, M., Tai, S.S., Lioyd, M., Modell, B., 1998. A multidisciplinary approach for improving service in primary care: randomized controlled trial of screening for hemoglobin disorders. Brit. Med. J. 317, 788–791. Muench, M.O., 2005. In utero transplantation: baby steps towards an effective therapy. Bone Marrow Transplant. 35, 537–547. Muench, M.O., Bárcena, A., 2004. Stem cell transplantation in the fetus. Cancer Control 11, 105–118. Owen, R.D., 1945. Immunologic consequences of vascular anastomoses between bovine twins. Science 102, 400–401. Peranteau, W.H., Flake, A.W., 2006. In-utero tolerance. Curr. Opin. Organ Transplant. 11, 353–359. Pixley, J.S., Tavassoli, M., Zanjani, E.D., Shaft, D.M., Futamachi, K.J., Sauter, T., Tavassoli, A., Mackintosh, F.R., 1994. Transplantation in utero of fetal human hematopoietic stem cells into mice results in hematopoietic chimerism. Pathobiology 62, 238–244. Rice, H.E., Hedrick, M.H., Flake, A.W., 1994. In utero transplantation of rat hematopoietic stem cells induces xenogeneic chimerism in mice. Transplant. Proc. 26, 126–128. Wang, M.L., Yan, J.B., Xiao, Y.P., Huang, S.Z., 2005. Construction of an allogenic chimeric mouse for the study of the behaviors of donor stem cells in vivo. Chin. Med. J. 118 (17), 1444–1450. Zanjani, E.D., Flake, A.W., Rice, H., Hedrick, M., Tavassoli, M., 1994. Long-term repopulating ability of xenogeneic transplanted human fetal liver hematopoietic stem cells in sheep. J. Clin. Invest. 93, 1051–1055. Zeng, Y.T., Huang, S.Z., 1985. α-Globin gene organization and prenatal diagnosis of αthalassemia in China. Lancet 1, 304–307. Zeng, F.Y., Chen, M.J., Katsumata, M., Huang, W., Gong, Z., Hu, W., Qian, H., Xiao, Y., Ren, Z., Huang, S., 2005. Identification and characterization of engrafted human cells in human/goat xenogeneic transplantation chimerism. DNA Cell Biol. 24, 403–409. Zeng, F.Y., Chen, M.J., Baldwin, D.A., Gong, Z.J., Yan, J.B., Qian, H., Wang, J., Jiang, X., Ren, Z.R., Sun, D., Huang, S.Z., 2006. Multiorgan engraftment and differentiation of human cord blood CD34+Lin− cells in goats assessed by gene expression profiling. Proc. Natl. Acad. Sci. U. S. A. 103, 7801–7806.
Contents lists available at ScienceDirect
Experimental and Molecular Pathology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x m p
Hematopoietic stem cell engraftment by early-stage in utero transplantation in a mouse model Xin Chen a,c, Xiu-li Gong a,c, Makoto Katsumata a,d, Yi-tao Zeng a,c, Shu-zhen Huang a,c,⁎, Fanyi Zeng a,b,c,⁎ a
Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Institute of Medical Science, Shanghai Jiao Tong University School of Medicine, Shanghai, China Key Laboratory of Embryo Molecular Biology, Ministry of Health of China, Shanghai, China d Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA b c
a r t i c l e
i n f o
Article history: Received 9 July 2009 Available online 7 August 2009 Keywords: In utero transplantation Hematopoietic stem cell Chimera establishment Allogeneic transplantation Fluorescence in situ hybridization
a b s t r a c t A novel intrauterine transplantation (IUT) approach was developed to improve the efficiency of engraftment of hematopoietic stem cells (HSCs). HSCs with a green fluorescent protein (GFP) reporter gene were transplanted in utero on days 12.5, 13.5 and 14.5 post coitum (p.c.). The degree of chimerism of donor cells in recipient newborn mice was examined using fluorescent microscopy, polymerase chain reaction (PCR), fluorescence-activated cell sorting (FACS), and fluorescence in situ hybridization (FISH) analyses. Microscopic examination revealed the presence of green fluorescent signal in the peripheral blood of the chimeric mice. The highest survival rate (47%) as well as the highest chimerism rate (73%) were achieved by our new approach in the newborn mice that were subjected to in utero transplantation (IUT) on day 12.5 p.c. (E12.5) compared to the conventional IUT method. FACS analysis indicated that 1.55 ± 1.10% of peripheral blood cells from the newborn mice were GFP-positive donor cells. FISH showed that cells containing the donor-specific GFP sequence were present in the bone marrow (BM) of the chimeric mice. Thus, the efficiency of chimera production with this new method of IUT was significantly improved over the existing IUT techniques and instruments. © 2009 Elsevier Inc. All rights reserved.
Introduction As a result of rapid advances in molecular and diagnostic medicine, a growing list of hereditary defects can be diagnosed before birth (Zeng and Huang, 1985; Modell et al., 1998; Flake and Zanjani, 1997). Prenatal diagnosis offers the opportunity for early management of those diseases. IUT of stem cells has been studied as one of the approaches for treating a range of genetic birth defects, including hemoglobinopathies, immunological defects and inborn errors of metabolism (Flake et al., 1996; Muench and Bárcena, 2004; Muench, 2005). However, prenatal treatment still faces many challenges due to poor understanding of stem cell behavior in vivo (Brännström and Wranning, 2007). Therefore, establishment of an effective animal
Abbreviations: IUT, in utero transplantation; HSC, hematopoietic stem cell; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; PCR, polymerase chain reaction; FACS, fluorescence-activated cell sorting; FISH, fluorescence in situ hybridization; BM, bone marrow; PBS, phosphate buffered saline; MNC, mononuclear cell; MACS, magnetic-activated cell sorting; PB, peripheral blood; RBC, red blood cell. ⁎ Corresponding authors. S. Huang is to be contacted at Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University, 24/1400 West Beijing Road, Shanghai 200040, China. Fax: +86 21 6247 5476. F. Zeng, Institute of Medical Science, Shanghai Jiao Tong University School of Medicine, 227 South Chongqing Road, Shanghai 200025, China. Fax: +86 21 6247 5476. E-mail addresses: [email protected] (S. Huang), [email protected] (F. Zeng). 0014-4800/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2009.07.009
model of IUT for the studies of homing, differentiation and proliferation of the donor stem cells would provide great insights into the prenatal treatment of many genetic diseases. In the past decade, IUT has been utilized in primates, sheep, goats, rats, and mice (Harrison et al., 1989; Zanjani et al., 1994; Zeng et al., 2005; Rice et al., 1994; Pixley et al., 1994). We have successfully established an experimental model of transplanting human HSCs into fetal goats using B-scan ultrasonography (Zeng et al., 2006). In the present study, we attempted to establish an IUT model in mice, which are suitable for studies of prenatal treatment because of their small size, short breeding cycle and defined genetic background. However, performing IUT in mice is much more challenging because the fetus is small and requires precise and skillful surgical techniques. As a result, the survival and chimeric rates are often very low. The purpose of this investigation is to establish an IUT procedure that can improve the success rate and achieve high levels of chimerism. To this end, we developed a new intrauterine transplantation instrument and modified existing surgical procedures. We assessed the improvement of the survival rate and degree of chimerism of engraftments of murine BM-derived HSCs by performing various molecular analyses. This new IUT methodology allows the efficient generation of chimeric mice that will provide a useful model for the studies of stem cell biology in vivo.
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Materials and methods Preparation of donor cells Donor cells were prepared from transgenic mice (4–8 months of age) obtained from the Shanghai Institute of Medical Genetics. The mice had an EGFP gene controlled by the β-globin promoter/ enhancer in which the EGFP (i.e., HS23-eGFP vector) is expressed in an erythroid-specific manner (Jia et al., 2003). Donor bone marrow (BM) was harvested sterilely by flushing the tibias and femurs with phosphate buffered saline (PBS) using a 26-gauge needle. A single cell suspension was prepared by 3 gentle passes through the needle. Cell suspensions were then filtered through a 70-μm nylon mesh and layered over Ficoll (1.077 g/ml; Axis-Shield PoC AS, Oslo, Norway). After centrifugation at 600 g for 20 min, the mononuclear cell (MNC) layer was removed and washed twice with PBS. Sca-1+ (mouse hematopoietic stem cell antigen) cells were enriched from MNCs by magnetic-activated cell sorting (MACS) with a Sca-1 progenitor cell isolation kit (Miltenyi Biotec, Bergish Gladbach, Germany) according to the manufacturer's instructions. The cells were counted prior to transplantation, and greater than 95% viability was confirmed by a trypan blue exclusion assay. Mice Mice of the Kun-Ming Bai strain (8–10 weeks old) were obtained from the animal facility of Shanghai Institute of Medical Genetics, Shanghai Children's Hospital; and their utilization was approved by the Review Board of Shanghai Children's Hospital. Fetuses of timed pregnant mice were transplanted on days 12.5 (E12.5), 13.5 (E13.5) and 14.5 (E14.5) p.c. (term gestation 21 days). Development of a new instrument for in utero transplantation A new instrument was designed as follows: a sterile plastic tube (Westingarea Corporation, China) was cut to 7–8 cm long. Its standard scalp interface end was connected to a 10 μl capacity adjustable pipettor; while the other end was connected to a 33-gauge injection needle (Popper & Sons, Germany). The injection volume was controlled via the pipettor (Supplementary Figs. 1B and C). Procedures for in utero transplantation
Fig. 1. In utero transplantation of murine Sca-1+ cells into fetal peritoneal cavity. The fetuses are injected using the novel injection instrument while the subject fetus is immobilized with a sterile cotton swab (A). The arrow indicates the injection site (B).
Identification of donor cell engraftment
Nested-PCR analysis DNA was extracted from blood cells of recipient mice. The GFP sequence was detected by a nested-PCR procedure using sequence specific primers (GFP primers: S1, 5′-TGACCTACGGCGTGCAGTGCTT3′; A1, 5′-TCGTCCATGCCGAGAGTGATCC-3′; S2, 5′-CACATGAAGCAGCACGACTTCT; and A2, 5′-AACTCCAGCAGGACCATGTGAT-3′). The amplification of sequence was as follows. In the first PCR, 15 μl of PCR mixture with the primers S1 and A1 was used. After the initial denaturing step at 94 °C for 5 min, 16 cycles of amplification were performed, including 45 s at 94 °C, 45 s at 58 °C, 1 min at 72 °C, and a final extension at 72 °C for 10 min. In the second PCR amplification, 25 μl of a mixture containing the primers S2 and A2 and 5 μL of the first PCR products was used. PCR conditions were similar to those of the first PCR except for the annealing step for 30 s. In total, 28 cycles were performed. As a control, β-actin primers were used (S, 5′CGCTCGTTGCCAATAGTGAT-3′; A, 5′-CCACAGGCATTGTGATGG-3′) with the annealing temperature at 60 °C for 28 cycles. The PCR products were separated in a 2.0% agarose gel and visualized by ethidium bromide staining.
Fluorescent cells analysis Blood samples (4–5 μl) were collected in heparinized microhematocrit tubes. Peripheral blood smears were prepared, air dried and fixed with 95% ethanol. GFP-red blood cells (RBCs) were identified using a Leica DM RXA2 fluorescent microscope.
Fluorescence-activated cell sorting (FACS) analysis FACS analysis was performed after birth at set intervals using approximately 10 μl of peripheral blood (PB) collected in heparinized capillary tubes via retro-orbital vein puncture of mice. The blood was then analyzed with flow cytometry to determine the presence of donor
Murine bone marrow-derived Sca-1+ cells were injected directly into the fetal peritoneal cavities. On E12.5, E13.5 and E14.5, pregnant mice were anesthetized with 1% pentobarbital sodium intraperitoneally (i.p.) at 8 ml/kg pre-pregnancy body weight. Using sterile technique, a midline laparotomy was made and the uterine horns were visualized. Each fetus was administered with 5 × 104 Sca-1+ cells i.p. in 3 μl of PBS through the intact uterine wall using the improved transplantation needle by a two-person operation, one responsible for the injection, and the other one for pipette control (Figs. 1A and B). Control animals received 3 μl PBS instead of the donor cells. The uterus was carefully placed back into the maternal peritoneal cavity, followed by abdominal closure. The mothers were kept warm until they recovered from anesthesia. The pups were carried to term, delivered normally, and subsequently weaned at 3 weeks of age.
X. Chen et al. / Experimental and Molecular Pathology 87 (2009) 173–177 Table 1 Survival and chimerism rates after IUT of murine Sca-1+ cells using the conventional vs. novel transplantation instruments. Group
No. of fetuses injected
No. of live-born pups
Survival rate (%)
Chimerism rate (%)a
Conventional Novel
81 73
28 44
34 60⁎
29 53⁎
The fetuses at day 14.5 of gestation were transplanted intraperitoneally with 5 × 10 murine BM-derived Sca-1+ cells. a Status of chimerism was confirmed by molecular biological methods. ⁎ p b 0.05 between the two groups.
4
GFP-RBCs. One hundred thousand cells were used per measurement, and duplicate measurements were performed for each sample. Fluorescence in situ hybridization (FISH) analysis The mouse BM was used for FISH analysis according to the methods described in our previous study (Zeng et al., 2005). To detect the donor GFP cell chimera, the HS23/EGFP vector was labeled with DIG-Nick Translation Mix (Roche, Germany) according to the manufacturer's protocol. FISH signals were examined with a Leica DM RXA2 fluorescent microscope. Images were captured with the photography system associated with the microscope.
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survival birth rate was significantly higher (60% vs. 34%, p b 0.05). Nested-PCR and FACS analyses were performed to verify the existence of donor cells in the peripheral circulation of the recipient mice. The rate of chimerism in the experimental group was significantly higher than that of the conventional group (53% vs. 29%, p b 0.05; Table 1). IUT of murine BM-derived Sca-1+ cells into the peritoneal cavities of fetal mouse recipients at different developmental stages We first examined the effect of IUT on stem cells as well as our technical consistency with the novel IUT instrument. Each recipient fetus received approximately 5 × 104 Sca-1+ cells in 3 μl of PBS at E12.5, E13.5 or E14.5 stage. A comparable number of control animals received the same volume of PBS. The survival rate of the Sca-1+ cell injected group at E12.5 was 47% (32/69), at E13.5 was 44% (27/62) and at E14.5 was 60% (44/73), while that of the control group was 49% (32/65), 49% (36/74) and 58% (39/68) at E12.5, E13.5 and E14.5, respectively. These survival rates were not significantly different between the two groups in each developmental stage (p N 0.05; Fig. 2A). The status of the engraftment of donor cells was examined by nested-PCR and FACS analyses. The results showed that the chimerism rate when we transplanted at E12.5 was 73%, which was significantly higher than that of E14.5 (53%; p b 0.05; Fig. 2B). Fluorescent microscopy analysis of peripheral GFP cells in chimeric mice
Statistical analysis All the data were presented by plotting the mean value and standard deviation of each group. Statistical comparisons between the groups were performed by chi-square test or two-tailed Student's t-test assuming unequal variances, and a p-value of b0.05 was considered significant.
The presence of donor GFP-RBCs in peripheral blood of the transplanted mice was confirmed by fluorescent microscopy. The results demonstrated that GFP-RBCs were present in the peripheral blood of transplanted mice but not in control mice. This illustrated that the hematopoietic stem cells of donor origin could differentiate into mature erythrocytes expressing GFP in transplanted mice (Fig. 3).
Results Nested-PCR analysis of donor genomic DNAs Construction of the transplantation instrument for IUT with murine BM-derived Sca-1+ cells The conventional instrument for IUT consists of a glass capillary (Supplementary Fig. 1A). In our injection instrument, the glass capillary was substituted with a 33-gauge injection needle to minimize the damage to the fetus. The amount of the HSCs to be injected was controlled by a pipette (Supplementary Figs. 1B and C). Both instruments were used for IUT on E14.5 fetuses. For the experimental group, we transplanted Sca-1+ cells into 73 fetuses in utero using the novel instrument, and 44 pups were born at term. In contrast to the group with the conventional injection method, the
Nested-PCR was carried out on newborn mice transplanted at 12.5 days of gestation to verify the existence or expansion of grafted donor cells in the peripheral circulation. Nineteen out of the twentysix transplanted mice showed donor GFP chimerism in their peripheral blood while the control group was negative (Fig. 4). FACS analysis of chimerism rate in recipients In order to confirm the stability of the donor cell engraftment, blood samples from the transplanted mice were examined by a series of flow cytometry analysis at 1, 4, 8 and 12 months of age. The average
Fig. 2. Comparison of survival and chimerism rates between murine Sca-1+ cell injected and control injected groups after IUT at different developmental stages. The newborn survival rates are not significantly different between the two groups (p N 0.05, n N 60) at each stage (A). The highest chimerism (73%) is achieved at E12.5, and this rate was significantly higher than that of E14.5 (p b 0.05, n N 60) (B).
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Fig. 3. Fluorescent microscope analysis of donor GFP cells in peripheral blood (PB) of transplanted mice (40×). TM = GFP transgenic mouse, SCT = transplanted mouse, NM = untransplanted mouse. Fluorescence only (A). Fluorescence + light (B). GFP-positive cells are identified in the PB of the transplanted mice, but not in the untransplanted mice.
proportion of donor GFP cells was 1.55% [mean ± SD = (1.55 ± 1.1)%] (Fig. 5). All the cells in peripheral blood from the control group of untransplanted mice were GFP negative. FISH analysis of cells with donor-specific GFP signals To further confirm the presence of donor cells, we performed interphase FISH experiments. A DNA probe specifically detected donor HS23/EGFP vector signals in BM cells of the HSC transplanted mice, but not in untransplanted mice (Fig. 6). Discussion Some cattle twins are dizygotic yet share placental circulation, a condition known as freemartinism. Persistent chimerism was documented in freemartin cattle, indicating that a significant exchange of cells occurred during development, which can result in the acquisition of donor-specific immune tolerance (Owen, 1945). The goal of fetal IUT is to mimic the effects of placental cross-circulation by directly transplanting cells into a fetus, thereby generating chimerism. In the IUT procedure, donor cells are transplanted by intraperitoneal (i.p.) injection. However, in the mouse experimental system, the IUT operation is rather difficult because the size of the fetuses is very small and there are multiple fetuses per pregnancy (regularly 6–10 fetuses). In the IUT procedure, a small glass needle is traditionally employed. The glass needles are intrinsically fragile, and it is cumbersome to adjust the injection volume using a syringe attached
Fig. 4. Detection of donor-specific GFP sequence in the transplanted mice by nestedPCR. Lane M: 100 bp DNA marker; lanes 1, 4–11: nine individually transplanted mice; lane 2: blank; lane 3: GFP transgenic mouse; lane 12: untransplanted mouse. Beta-actin DNA was amplified as an internal control.
at the other end of the needle (Wang et al., 2005). As a result, the process is often unnecessarily prolonged, resulting in increased risk of fetal death and later abortion. To circumvent these problems, we systematically changed the design of the injection instrument and operational scheme. We utilized the smallest sterile disposable injection needle (33-gauge) to minimize the damage to the fetuses. Then, we connected a 10 μl capacity adjustable pipettor to the injection needle so that the total volume to be injected could be accurately controlled by adjusting the pipettor. We also shortened the total time necessary for a series of injections for all fetuses to less than 10 min by the two-person operation mentioned in the Materials and methods section. The efficiency of the IUT appears to be significantly improved with these modifications. IUT is a traumatic operation compared with some other surgeries, and a couple of important technical details should be noted for the success of this procedure. Close monitoring of the anesthesia state is essential in addition to the choice of the anesthetics and doses. In the pregnant mice, the fetuses account for a large part of the weight. The initial dose of anesthetic should be calculated based on the prepregnancy weight of the recipient mice, and depth of the anesthesia should also be monitored periodically throughout the procedure by pinching toes or observing the breathing rate. Additional anesthetic
Fig. 5. FACS analysis of donor cell engraftment in recipient mice at 1, 4, 8 and 12 months of age. Nineteen different dots represent proportion of donor GFP cells in nineteen individually transplanted mice. No GFP cells were detectable in untransplanted mice.
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Fig. 6. Fluorescence in situ hybridization (FISH) of the BM cells of representative mice. TM = GFP transgenic mouse. Each of the BM cells at interphase shows one hybridization signal. SCT = transplanted mouse. One of the BM cells in the field shows hybridization signal. NG = untransplanted mouse. None of the BM cells examined shows hybridization signal.
needs to be administered as necessary. Routine preoperative care, e.g. administration of analgesia and aseptic surgical environment, is also important. The other factor is the intra- or postoperative care. Close monitoring and maintenance of normal body temperature of the recipient mouse during and after surgery are important to prevent abortion. Providing a clean and quiet housing environment, highenergy diet with supplements and administration of an analgesic are also important for successful pregnancy. Our current investigation clearly showed that the developmental stage at which IUT is performed on the fetal recipients is the most important factor for a successful engraftment of HSCs. Pregnancy is divided into three stages; namely, early, medium and late periods. For most large animals, IUT is performed at the early period when the immune system of the fetuses is immature and the donor stem cells are easier to engraft in the host. However, in the mouse, IUT has been performed at the mid period (typically at E14.5) because the basic form of the fetuses at the early stage (before E7) is too small to distinguish (Hayashi et al., 2002; Frattini et al., 2005). In our current experiment, we were able to perform IUT at E12.5 for the first time. When the efficiency of the IUT in terms of survival rate and the degree of chimerism was compared among fetuses subjected to IUT at E12.5, E13.5 and E14.5, it is important to note that while the survival rates did not decrease at all for fetuses with IUT at E12.5, the degree of chimerism at this stage was the highest. Fetal immunologic tolerance might have played an important role for this improvement in relation to the degree of fetal thymic development (Peranteau and Flake, 2006). Thus, by not compromising the survival of the embryos at this earlier developmental stage, we significantly improved the IUT efficiency by producing a higher degree of chimerism. We successfully verified this by fluorescent microscopy, nested-PCR, and FACS, as well as FISH analyses. It should be noted that our technological advancement in designing the novel injection instrument and scheme contributed to the creation of mice with higher levels of allogeneic chimerism following IUT. In conclusion, we successfully modified and improved the IUT instruments and procedures for efficient allogeneic transplantation with a high success rate and degree of chimerism in order to study the biological properties of hematopoietic stem cells in vivo in a mouse model. Intrauterine stem cell transplantation provides a great potential advantage in prenatal treatment. In the near future, with the in-depth study of stem cell biology, IUT could be routinely applied in clinical treatments of many genetic defects. Acknowledgments This work was supported by grants from the National Key Scientific Research Program of China (2007CB947800 to FZ) and National Basic Research Program of China (2007CB511904 to SH). We especially thank Professor Hao Shen for his helpful discussion for this study. We also thank Mr. Yin Cheng for his excellent technical support.
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