Improved Safety of Hematopoietic Transplantation with Monkey Embryonic Stem Cell



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江边孤钓

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发表于 2009-3-5 00:05 |显示全部帖子

作者：Hiroaki Shibataa,b, Naohide Ageyamab, Yujiro Tanakaa, Yukiko Kishia, Kyoko Sasakia, Shinichiro Nakamurab,c, Shin-ichi Muramatsud, Satoshi Hayashie, Yoshihiro Kitanof, Keiji Teraob, Yutaka Hanazonoa作者单位：a Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan;b Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Ibaraki, Japan;c Department of Veterinary Pathology, Nippon Veterinary and Animal Science University, Tokyo, Ja
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      【摘要】
   Cynomolgus monkey embryonic stem cell (cyESC)-derived in vivo hematopoiesis was examined in an allogeneic transplantation model. cyESCs were induced to differentiate into the putative hematopoietic precursors in vitro, and the cells were transplanted into the fetal cynomolgus liver at approximately the end of the first trimester (n = 3). Although cyESC-derived hematopoietic colony-forming cells were detected in the newborns (4.1%¨C4.7%), a teratoma developed in all newborns. The risk of tumor formation was high in this allogeneic transplantation model, given that tumors were hardly observed in immunodeficient mice or fetal sheep that had been xeno-transplanted with the same cyESC derivatives. It turned out that the cyESC-derived donor cells included a residual undifferentiated fraction positive for stage-specific embryonic antigen (SSEA)-4 (38.2% ¡À 10.3%) despite the rigorous differentiation culture. When an SSEA-4-negative fraction was transplanted (n = 6), the teratoma was no longer observed, whereas the cyESC-derived hematopoietic engraftment was unperturbed (2.3%¨C5.0%). SSEA-4 is therefore a clinically relevant pluripotency marker of primate embryonic stem cells (ESCs). Purging pluripotent cells with this surface marker would be a promising method of producing clinical progenitor cell preparations using human ESCs. 7 }6 T- ?' l0 P' C) p1 g0 {: p' B
      【关键词】 Cynomolgus monkey Hematopoiesis Embryonic stem cell In utero transplantation Teratoma Purging Tumor prevention  h/ B2 X- E" ]" C5 i
   INTRODUCTION7 I& I/ }2 o7 I$ W3 @3 S. a( L5 x5 e

Human embryonic stem cells (hESCs) hold great potential in the treatment of a variety of diseases and injuries because embryonic stem cells (ESCs) have the ability to proliferate indefinitely in culture and to differentiate into any cell type .
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We have also reported a novel method for hematopoietic engraftment from cyESCs in sheep . In the present study, we have examined the safety as well as the efficacy of hematopoietic engraftment of cells derived from cyESCs in the allogeneic transplantation model.; P. T" y* E. F/ y5 s1 {
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MATERIALS AND METHODS
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Animals( [2 n. i; V0 ?& m8 W9 L" H
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Pregnant cynomolgus monkeys (16¨C22 years old) were obtained by mating and were reared at the Tsukuba Primate Research Center in accordance with Rules for Animals Care and Management set forth by the Research Center and Guiding Principles for Animal Experiments Using Nonhuman Primates formulated by the Primate Society of Japan. Experimental procedures were approved by the Animal Welfare and Animal Care Committee of the National Institute of Infectious Diseases. The animals were free of intestinal parasites and were seronegative for herpes virus B, varicella-zoster-like virus, measles virus, and simian immunodeficiency virus.
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Cell Preparation& h2 _0 ?, U5 y# Z4 J# I+ H

A cyESC line (CMK6G) stably expressing green fluorescent protein (GFP) was established after transfection of the parental cyESC line (CMK6) with the enhanced GFP gene (Clontech, Palo Alto, CA, http://www.clontech.com) .' R" ?2 v7 S% q- N
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cyESCs were induced to differentiate into the putative hematopoietic precursors as previously described . Briefly, undifferentiated cyESCs were transferred onto mitomycin C-treated confluent OP9 cells and cultured for 6 days in Iscove¡¯s modified Dulbecco¡¯s medium (Invitrogen) supplemented with 8% FCS, 8% horse serum (Invitrogen), 5 x 10¨C6 M hydrocortisone (Sigma, St. Louis, http://www.sigmaaldrich.com), and multiple cytokines, including 20 ng/ml recombinant human (rh) bone morphogenetic protein-4 (R&D Systems, Minneapolis, http://www.rndsystems.com), 20 ng/ml rh stem cell factor (Biosource, Camarillo, CA, http://www.biosource.com), 20 ng/ml rh vascular endothelial growth factor (VEGF; R&D Systems), 20 ng/ml rh Flt-3 ligand (PeproTech, Rocky Hill, NJ, http://www.peprotech.com), 20 ng/ml rh interleukin-3 (PeproTech), 10 ng/ml rh interleukin-6 (PeproTech), 20 ng/ml rh granulocyte colony-stimulating factor (PeproTech), and 2 IU/ml rh erythropoietin (Roche, Basel, Switzerland, http://www.roche.com). The cells were resuspended in 0.1% human serum albumin (Sigma)/Hanks¡¯ balanced saline solution (Sigma) for transplantation.* y8 H9 D, Z8 K1 E& g
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Flow Cytometry$ k0 ?+ S* Q; o
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Primary antibodies (Abs) used in the present study were anti-human CD34 monoclonal Ab (mAb; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen), anti-human CD31 mAb (Pharmingen), anti-human CD45 mAb (Pharmingen), anti-human vascular endothelial (VE) cadherin mAb (Pharmingen), rabbit anti-human VEGF receptor (VEGFR)-2 Ab (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), and anti-stage-specific embryonic antigen (SSEA)-4 mAb (Chemicon, Temecula, CA, http://www.chemicon.com). All of them cross-reacted to cynomolgus counterparts as previously demonstrated . Secondary Abs were phycoerythrin (PE)-conjugated rabbit anti-mouse immunoglobulins (Ig) Ab (DakoCytomation, Glostrup, Denmark, http://www.dako.com) and Alexa Fluor 647-conjugated goat anti-mouse IgG Ab (Molecular Probes, Eugene, OR, http://probes.invitrogen.com). Cells stained with unlabeled primary Abs were incubated with fluorescence-labeled secondary Abs. Cells were incubated with either primary or secondary Ab for 20¨C60 minutes at 4¡ãC. Regarding staining with the anti-VEGFR-2 Ab, the cells were incubated with biotin-conjugated goat anti-rabbit IgG Ab (Beckman Coulter, Miami, http://www.beckmancoulter.com), followed by PE-conjugated streptavidin (Beckman Coulter). Fluorescence-labeled cells were analyzed with a FACS Calibur flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Data analysis was performed using the CellQuest software (Becton, Dickinson and Company). Isotype-matched, irrelevant mAbs (DakoCytomation or Beckman Coulter) served as negative controls. Nonviable cells were excluded from analysis by propidium iodide (Sigma) costaining.
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Cell Sorting

Cell sorting was performed to purge SSEA-4  cells from among the cultured cyESCs in vitro. Cells were incubated with the anti-SSEA-4 mAb for 1 hour at 4¡ãC and washed twice with Dulbecco¡¯s modified Eagle¡¯s medium supplemented with 10% FCS. The cells were then incubated with the PE-conjugated anti-mouse Ig Ab for 1 hour at 4¡ãC and washed twice again. GFP-positive and SSEA-4-negative cells were sorted using an Epics Elite cell sorter (Beckman Coulter). Data acquisition was performed using the Expo2 software (Beckman Coulter).

Transplantation and Delivery
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Transplant procedures were previously described . Briefly, animals were anesthetized via an intramuscular administration of ketamine hydrochloride (Ketalar, 10 mg/kg; Sankyo, Tokyo, http://www.sankyo.co.jp) and received 0.5%¨C1.0% isoflurane by inhalation by means of an endotracheal tube. Cells (0.16¨C46 x 106 cells per fetus; Table 1) were injected into the fetal liver through a 23-gauge needle using an ultrasound-guided technique at approximately the end of the first trimester. The fetuses were delivered by cesarean section at 2¨C3 months after transplant (gestation 120¨C157 days, full term 165 days).
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Table 1. ESC-derived hematopoiesis and tumor formation
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Colony Polymerase Chain Reaction% V4 ]! P. G1 n* Z8 ^- S. j

Cynomolgus clonogenic hematopoietic colonies were produced as previously described . After cells were cultured in methylcellulose medium for 10¨C14 days, well-separated individual colonies were plucked into 50 µl of distilled water and digested with 20 µg/ml proteinase K (Takara, Shiga, Japan, http://www.takara-bio.com) at 55¡ãC for 1 hour, followed by 99¡ãC for 10 minutes. Each sample (5 µl) was used for a nested polymerase chain reaction (PCR) to detect the GFP gene sequence. The outer primer set was 5'-AAGGACGACGGCAACTACAA-3' and 5'-ACTGGGTGCTCAGGTAGTGG-3', and the inner primer set was 5'-GCATCGACTTCAAGGAGGAC-3' and 5'-GTTGTGGCGGATCTTGAAGT-3'. Amplification conditions for both the outer and inner PCR were 30 cycles of 95¡ãC for 30 seconds, 65¡ãC for 30 seconds, and 72¡ãC for 30 seconds. The outer PCR products were purified using a QIA quick PCR purification kit (Qiagen, Valencia, CA, http://www.qiagen.com). Simultaneous PCR for the ß-actin sequence was also performed to ensure DNA amplification of the sample in each colony. The primer set for ß-actin was 5'-CATTGTCATG-GACTCTGGCGACGG-3' and 5'-CATCTCCTGCTCGAAG-TCTAGGGC-3'. Amplification conditions for ß-actin PCR were 40 cycles of 95¡ãC for 30 seconds, 65¡ãC for 30 seconds, and 72¡ãC for 30 seconds. Amplified GFP (131 bp) and ß-actin (234 bp) products were resolved on 2% agarose gel (Sigma) and visualized by ethidium bromide (Invitrogen) staining.

RNA PCR

Total RNA was extracted from cells of interest using the EZ1 RNA universal tissue kit (Qiagen). RNA was reverse-transcribed at 50¡ãC for 30 minutes using the RNA LA PCR kit (Takara) with oligo dT primer. The resulting cDNA was then subjected to PCR. Regarding PCR for Oct-4, the primer set was 5'-GGACACCTGGCTTCGGATT-3' and 5'-TTCGCTTTCTC-TTTCGGGC-3'. The PCR conditions were 35 cycles of 95¡ãC for 30 seconds, 67¡ãC for 45 seconds, and 68¡ãC for 1.5 minutes. Regarding PCR for Scl, the primer set was 5'-GGGCG-GAAAGCTGTTTGCGATT-3' and 5'-TCGCTGAGAGGCCT-GCAGTT-3'. The PCR conditions were 35 cycles of 95¡ãC for 30 seconds, 63¡ãC for 1 minute, and 72¡ãC for 1 minute. A simultaneous PCR for ß-actin was also conducted on each cDNA sample as an internal control as described above. Amplified Oct-4 (697 bp), Scl (201 bp), and ß-actin (234 bp) products were resolved on 2% agarose gel and visualized by ethidium bromide staining.
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RESULTS) ]1 c- s2 m1 b6 W' K9 r
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In Utero Transplantation and Delivery3 n% D) ?( _( }# y  I. w
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cyESCs stably expressing GFP were used in this study . The GFP expression was stable during the 6-day culture (Fig. 1A, 1B) and afterward (data not shown).
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Figure 1. Flow cytometric analysis during the in vitro differentiation of cyESCs. Undifferentiated cyESCs expressing green fluorescent protein were cultured on OP9 cells with multiple cytokines (see Materials and Methods). (A): Cells on day 0 are shown in bright (left) and dark (right) fields. (B): Cells on day 6 are shown in bright (left) and dark (right) fields. (C): Cells on days 0, 4, 6, 12, and 18 were stained for CD34. (D): Cells on days 0, 4, 6, 12, and 18 were stained for CD31. (E): Cells on days 0, 4, 6, 12, and 18 were stained for VE-cadherin. (F): Cells on days 0, 4, 6, 12, and 18 were stained for CD45. The vertical axis shows the fraction (percentage) of cells that were stained positive. (C¨CF): Results of two or three independent experiments are shown. (G): Although cells on day 0 already express low levels of VEGFR-2, a VEGFR-2high population did not emerge until day 6. (H): Dot-plot profiles for VEGFR-2 and VE-cadherin expression indicate that cells positive for both VEGFR-2 and VE-cadherin emerged until day 6. (G, H): Representative results from three independent experiments are shown. (I): The Scl gene expression was upregulated on day 6 to a level similar to that in the cynomolgus fetal liver as assessed by RNA polymerase chain reaction. Day-6 cells (putative hematopoietic precursors) were used for transplantation. Abbreviations: cyESC, cynomolgus embryonic stem cell; cyFL, cynomolgus fetal liver; DW, distilled water; VE, vascular endothelial; VEGFR, vascular endothelial growth factor receptor./ U; |0 ~" A" U" H% g3 j
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Teratoma Formation) W' p) N9 T& v  t% [+ |( J( |
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The undifferentiated cyESCs (n = 3) or cyESC-derived putative hematopoietic precursors (n = 3) were transplanted in utero into allogeneic fetuses in the liver under ultrasound guidance at approximately the end of the first trimester (49¨C66 days, full term 165 days) (Table 1). Regardless of whether the undifferentiated cyESCs or putative hematopoietic precursors were transplanted, tumors were found in the thoracic or abdominal cavities in all the six animals at 2¨C3 months after transplant (Table 1; Fig. 2A). The tumors fluoresced (Fig. 2B) and consisted of three germ layer cells. Thus, they were teratomas derived from transplanted cells. However, tumors were hardly observed in fetal sheep (1/10;  and our unpublished data) (Table 1) and immunodeficient (nonobese diabetic/severe combined immunodeficient) mice (3/10; our unpublished data) after the same putative hematopoietic precursors were transplanted.
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Figure 2. Tumor formation after the transplantation of cynomolgus embryonic stem cell (cyESC)-derived progenitor cells. Tumors formed in all three monkey fetuses transplanted with the day-6 cyESC-derived progenitor cells (putative hematopoietic precursors). (A): A representative tumor in the thoracic cavity at 3 months after transplantation (monkey no. 0841). (B): The tumor was observed in bright (left) and dark (right) fields under a fluorescence microscope.

In Vivo cyESC-Derived Hematopoiesis: K/ T" X; a& _+ Z4 c1 l8 m
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Regarding the newborn monkeys that had been transplanted with the putative hematopoietic precursors, we harvested cells from the femur, cord blood, and liver and plated the cells in methylcellulose medium to produce clonogenic hematopoietic colonies (colony-forming units ., }4 b4 }0 L9 q8 K: M" x

Figure 3. cyESC-derived hematopoiesis in vivo. (A): Bone marrow, cord blood, and liver cells were harvested from newborn monkeys and placed in methylcellulose medium to produce clonogenic hematopoietic colonies. (B): A cytospin specimen (stained with the May-Giemsa method) of plucked colonies reveals mature neutrophils. To identify cyESC-derived colonies, well-separated individual colonies were plucked and examined for the GFP sequence by PCR. Plucked MeC alone (not containing colonies) served as a negative control. PCR of the ß-actin sequence in the same colonies was simultaneously performed as an internal control. Colony PCR was repeated at least twice. (C): Representative colony PCR results for monkey no. 0021. Asterisk indicates bands positive for the GFP sequence. Abbreviations: CMK6G, positive control green fluorescent protein-expressing cynomolgus cells; cyESC, cynomolgus embryonic stem cell; DW, distilled water; GFP, green fluorescent protein; M, molecular weight marker; MeC, methyl-cellulose; PCR, polymerase chain reaction.

Purging SSEA-4  Cells of the Putative Hematopoietic Precursors1 Z( c' J6 g. u. ~# F4 x8 B

We examined the expression of an undifferentiated primate ESC marker, SSEA-4, in the undifferentiated cyESCs (day 0) and putative hematopoietic precursors (day 6). The proportion of SSEA-4  cells was 93.4% ¡À 8.1% and 38.2% ¡À 10.3% among the day-0 and -6 cells, respectively (Fig. 4A). A substantial number of cells were still positive for SSEA-4 after the rigorous differentiation culture. In addition, a considerable number of cells expressing another undifferentiated marker, Oct-4, remained among the day-6 population as assessed by RNA-PCR (Fig. 4B). Those residual undifferentiated cells might be responsible for the formation of teratomas in the recipients.

Figure 4. Purging SSEA-4  cells from among cyESC-derived progenitor cells. (A): Undifferentiated cyESCs (day 0) and cyESC-derivatives (day 6) were stained with anti-SSEA-4. The SSEA-4 expression (percentage of total) at day 0 and day 6 is shown (n = 8). (B): The Oct-4 expression at days 0 and 6 was also examined by RNA polymerase chain reaction. (C): Flow cytometric dot-plot profiles are shown for the SSEA-4 versus GFP expression at day 0 (left), at day 6 before the purge (middle), and at day 6 after the purge (right). Six independent experiments were conducted, and similar results were obtained. (D): No tumors were detected in any monkey after the transplantation of SSEA-4-negative day-6 cyESC derivatives (a representative monkey, no. 0981). Abbreviations: cyESC, cynomolgus embryonic stem cell; GFP, green fluorescent protein; M, molecular weight marker; SSEA, stage-specific embryonic antigen.2 d! u4 @4 Y8 E) Y/ P3 O6 E: x
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To prevent teratomas from forming in recipients, we purged SSEA-4  cells of the putative hematopoietic precursors and transplanted the SSEA-4¨C population into the fetal monkey liver (n = 6) (Fig. 4C). At delivery, tumors were no longer observed in the six animals that had been transplanted with the sorted SSEA-4¨C cells (Fig. 4D). The transplanted cell-derived CFU were clearly detected in the newborns, and the fraction was not spoiled (2.3%¨C5.0%; Table 1), although the removed SSEA-4  fraction included some CD34  cells (data not shown).' w' y2 f* o5 t# U( t

DISCUSSION& F! T% G/ w( M8 ^2 v3 H( _  i. R. c

We have previously described a method for hematopoietic engraftment from cyESCs ) (Table 1). To enhance ESC-derived hematopoiesis, further consideration is required of the in vitro culture conditions (i.e., the cytokine milieu, coculture- or embryoid body-associated cellular microenvironment, culture period, and genetic manipulation) and the in utero transplantation conditions (i.e., the preconditioning, route, and timing).8 T1 x: W' j: ^5 b$ T/ S

Teratomas developed in all animals, even after the transplantation of ESC-derived progenitor cells that had been cultured for 6 days in the differentiation medium. The risk of tumor formation was high, given that we could hardly detect tumors in immunodeficient mice or fetal sheep that had been transplanted with the same day-6 cyESC derivatives (. Our monkey allogeneic transplantation setting would therefore allow the strict evaluation of the in vivo safety of transplantation therapies using ESCs. However, given that teratomas indeed form when undifferentiated cyESCs alone are xeno-transplanted into immunodeficient mice, it is unclear why residual undifferentiated cells included among the day-6 cyESC derivatives did not form teratomas in immunodeficient mice or fetal sheep.* _; Y, h3 W- O3 S
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SSEAs that are developmentally regulated during early embryogenesis are widely used as markers to monitor the differentiation of both mouse and human embryos and ESCs . They found that the expression of Oct-4 is colo-calized with that of prostate apoptosis response-4, a protein mediating ceramide-induced apoptosis. Treatment of ESC-derived neural precursors with ceramide resulted in selective elimination of residual Oct-4-positive pluripotent cells. Our method, however, uses a cell surface marker to purge pluripotent cells. With this method, one can see the purging efficiency in real-time. This would be meritorious for clinical applications. Although we used a cell sorter to obtain the SSEA-4¨C fraction in the present study, selection with beads would be easier and more appropriate for clinical applications.
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To generalize the use of SSEA-4 for eliminating undifferentiated cells from among donor cells, we differentiated cyESCs into neural stem cells. After the culture, approximately 10% of cells were still positive for SSEA-4. When all the cells were transplanted into the striatum of Parkinson¡¯s cynomolgus monkeys, teratomas developed. We then transplanted cyESC-derived neural stem cells without an SSEA-4  fraction into the cynomolgus striatum and successfully detected the engraftment without tumor formation (our unpublished data). The removal of SSEA-4  cells is useful at least for hematopoietic and neural lineages.% |' `( q6 n5 \) X0 y/ g9 }) H4 n
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CONCLUSION
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We are now able to prevent the formation of tumors in nonhuman primate recipients by purging SSEA-4  cells from among ESC-derived progenitor cells without spoiling the engraftment. SSEA-4 is therefore a clinically relevant pluripotency marker of primate ESCs. Purging pluripotent cells with this marker would be a promising method for producing clinical progenitor cell preparations using hESCs to improve safety in vivo.3 C- _. D6 c0 T# t2 V$ K! |
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DISCLOSURES

The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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We thank Norio Nakatsuji (Kyoto University, Kyoto, Japan) and Yasushi Kondo (Tanabe Seiyaku Co., Ltd., Osaka, Japan) for providing cyESCs; Toru Nakano (Osaka University, Osaka, Japan) for providing OP9 cells; and Naomi Terao and Naomi Takino for technical assistance. This study was supported by grants (JMS 21st Century COE program, High-tech Research Center program, and Creation of Innovations) from the Ministry of Education, Culture, Sports, Science and Technology of Japan as well as grants (KAKENHI) from the Ministry of Health, Labor and Welfare of Japan.. O9 F5 @0 m( S# Q6 p
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