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EMBRYONIC STEM CELLS |
aDivision of Immunotherapy, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA;
bFirst Department of Pathology, Transplantation Center, Kansai Medical University, Osaka, Japan;
cPathology Core Facility, Northwestern University Cancer Center, Chicago, Illinois, USA;
dNewLink Genetics Corporation, Iowa State University Research Park, Ames, Iowa, USA
Key Words. Embryonic stem cells • Diabetes mellitus • Immune tolerance • Hematopoiesis
Correspondence: Correspondence: Richard K. Burt, M.D., 750 North Lake Shore Drive, ABA Building 6-649, Chicago, Illinois 60611, USA. Telephone: 312-908-0059; Fax: 312-908-0064; e-mail: rburt{at}northwestern.edu
Received on April 28, 2006;
accepted for publication on October 19, 2007.
First published online in STEM CELLS EXPRESS November 1, 2007.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...References |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...References |
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For leukemia, the lower relapse rate and higher percentage of cure following allogeneic HSCT, referred to as a graft-versus-leukemia (GVL) effect, is not clinically separable from GVHD [5]. Since both GVL and GVHD arise from donor lymphocytes, suppression or prevention of GVHD through anergy or deletion of donor lymphocytes also inhibits or prevents GVL. In autoimmune diseases, the beneficial effect of allogeneic HSCT, theoretically mediated by infused donor lymphocytes, was termed graft versus autoimmunity (GVA) [6, 7]. To determine whether donor allogeneic hematopoietic stem cells (HSC) independent of infused donor lymphocytes can confer a beneficial GVA effect that can be separated from and is independent of donor lymphocyte-mediated GVHD, we performed transplants in NOD mice using embryonic stem cell (ESC)-derived HSC, an ex vivo-derived stem cell source that is not contaminated with donor immune cells.
ESC are derived from the inner cell mass of the blastocyst (preimplantation embryo) and are pluripotent; that is, they can differentiate into cells of all three germ layers (mesoderm, ectoderm, and endoderm) [8]. Due to their multi-germ line plasticity, without further ex vivo manipulation, ESC transplants are prone to in vivo formation of teratomas [9, 10]. To prevent teratoma formation, ESC may be differentiated ex vivo into multipotent lineage-restricted adult hematopoietic stem cells, which are designated ESC-derived HSC [10, 11]. These ESC-derived HSC may be used as an alternate marrow donor source that can engraft across major histocompatibility complex (MHC) barriers without any adverse consequences, such as GVHD or teratoma formation [10]. Embryonic stem cells have been the topic of much professional and lay literature, yet despite their potential, only limited data are available on the in vivo application of ESC to cure disease [12, 13]. Herein, we demonstrate that ex vivo differentiation of ESC into adult hematopoietic progenitor cell phenotype prior to in vivo application confers a GVA effect manifest as islet cell tolerance without GVHD, despite crossing MHC barriers and absence of post-transplant immune suppression.
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MATERIALS AND METHODS |
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Induction of ESC Toward Hematopoietic Progenitors (HSC)
To induce differentiation toward HSC in vitro, embryonic stem cells were cultured on low-adherent Petri dishes in Iscove's modified Dulbecco's medium containing approximately 1% methylcellulose, 15% FBS, 150 µM monothioglycerol, 2 mM L-glutamine, 500 ng/ml murine stem cell factor, 46 ng/ml murine interleukin-3, and 500 ng/ml human interleukin-6 (StemCell Technologies and Sigma-Aldrich [St. Louis, http://www.sigmaaldrich.com]). Cells were cultured at 37°C in a 5% CO2 atmosphere incubator for 9–11 days.
Selection of Hematopoietic Progenitor Cells
Single-cell suspensions of cells differentiated from embryonic stem cells were collected, washed, suspended in phosphate-buffered saline (PBS), and sorted with anti-c-kit (CD117) antibody using OctoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) according to the manufacturer's protocol. The purity of selection was >90%. Following sorting, cells were resuspended in PBS and used immediately for injection.
Flow Cytometric Analysis
Two- or three-color flow cytometric analysis was performed using standard procedures on an Epics XL flow cytometer (Beckman Coulter, Miami, FL, USA). Single-cell suspensions were aliquoted and stained with either isotype controls or antigen-specific antibodies. Cell surface antigens were labeled with combinations of the following fluorescein isothiocyanate (FITC)-, or phycoerythrin (PE)-conjugated monoclonal antibodies: CD117 (c-kit), CD45/B220, CD19, CD11b, CD3, CD4, and CD8 (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml). Dead cells were excluded from analysis using propidium iodide staining. Samples were run on an Epics XL flow cytometer and analyzed with CellQuest software (BD Immunocytometry Systems, San Jose, CA, http://www.bdbiosciences.com/immunocytometry-systems/products) [10].
Mice
Six-week-old female NOD/LtJ (Kd, I-Ag7, Db) mice were purchased from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org) and used as recipients of ESC-derived hematopoietic progenitors (originated from 129/SvJX129/SV-CP male F1 hybrid 3.5-day-old mouse ESC H-2b). Mice were subjected to total body irradiation (TBI) at 8.0 Gy (in two doses) 16 hours before ESC-derived HSC injection. Mice were housed in microisolator cages under specific pathogen-free conditions and provided with 
-irradiated food in the animal facilities of Northwestern University. All animal experiments were approved by the Institutional Animal Care and Use Committee of Northwestern University.
Transplantation
Cells were prepared as described above and injected either intravenously (i.v.) or intra-bone marrow (IBM). For i.v. injection, 5 x 106 ESC-derived HSC in 0.25 ml were injected into one of the lateral tail veins. For IBM injection, 1 x 106 cells in 15 µl were injected into the tibia marrow cavity as previously described [10, 14].
Blood Glucose Level Measurements
A drop of peripheral blood was obtained by puncturing one lateral tail vein with an insulin needle. Blood glucose was measured using OneTouch Strip and Accu-Check Advantage Kit (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). Measurements were performed twice a week after mice achieved the age of 14 weeks or if they developed symptoms such as polyuria, polydipsia, or weight loss. Glycemia above 300 mg/ml was considered hyperglycemic. Mice were sacrificed upon a diagnosis of diabetes, which was defined as the presence of symptoms as described above and hyperglycemia in two consecutive measurements, or hyperglycemia in three consecutive blood glucose measurements.
Chimerism
The presence of ESC-derived (H-2Kb) chimerism was determined using flow cytometric analysis of peripheral blood 10, 24, and 40 weeks after ESC-derived HSCT and splenocytes 40 weeks post-transplantation or at the time of euthanasia. Cell surface antigens were labeled with the following FITC-, PE-, or biotin-conjugated monoclonal antibodies: H-2Kb, H-2Kd, CD45, CD3, CD19, CD4, and CD8 and isotype controls (BD Pharmingen) using standard procedure. Mononuclear cells isolated from the peripheral blood or spleen of an untreated NOD mouse (H-2Kd) were used as a negative control, whereas mononuclear cells from a 129/Sv mouse served as a positive control.
Glutamic Acid Decarboxylase Form 65-Induced T-Cell Response Assay
Spleen cells were plated at 5 x 105 nucleated cells in 100 µl of medium per well in 96-well flat-bottomed plates. Cells were cultured in RPMI 1640 (Cellgro; Mediatec, Washington, DC, http://www.cellgro.com) supplemented with 10% FBS, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 100 U/ml penicillin, and 50 µg/ml gentamicin. Glutamic acid decarboxylase form 65 (GAD65) (Diamyd Medical, Akron, OH, http://www.diamyd.se) was used as pancreatic β cell antigen. Peptides (GAD65 or control antigens including human serum albumin and murine myelin basic protein) were added to a final concentration of 20 µg/ml. Bromodeoxyuridine (BrdU) was added for the last 24 hours of culture. Supernatants were collected after 48 and 72 hours of antigenic stimulation and tested for the presence of interferon (IFN) by enzyme-linked immunosorbent assay (ELISA) using an IFN ELISA Kit (BD Pharmingen) according to the manufacturer's protocol.
Mixed Lymphocyte Reaction In Vitro
Immune responses in recipient NOD mice toward donor histocompatibility antigen of 129/Sv strain (ESC strain), recipient MHC (NOD mice), and third-party (BALB/c) antigens were evaluated by mixed lymphocyte reaction (MLR) tests. MLR tests were performed in six animals transplanted with ESC-derived cells, 40 weeks after transplantation. Splenocytes (1 x 106) from chimeric mice were cultured separately in 24-well plates (Falcon, BD Labware, Franklin Lakes, NJ, http://www.bdbiosciences.com), with irradiated splenocytes (30 Gy) (1 x 106) obtained from BALB/c and 129/Sv mice. The cells were cultured in a total volume of 2 ml of medium, as described above. Seventy-two hours later, cells were pulsed with BrdU by adding 20 µg of BrdU per 2-ml well. Twenty hours after the BrdU pulse, cells were harvested and processed with a BrdU Flow Kit (BD Pharmingen) according to the manufacturer's protocol. Cells were stained with FITC anti-BrdU and 7-aminoactinomycin. Flow cytometric data were acquired using an Epics XL flow cytometer and analyzed with CellQuest software. Syngeneic splenocytes and allogeneic splenocytes were used as negative and positive controls, respectively.
Histology
Histologic analysis was performed blindly (S. Ikehara, Osaka, Japan) on the native pancreas after the onset of overt diabetes or after planned euthanasia of normoglycemic animals. Pancreata were fixed in 4% buffered formalin. Hematoxylin-eosin and immunohistochemical staining were performed on paraffin-embedded tissue sections using standard procedures.
Fluorescence In Situ Hybridization Analysis
Single-label (whole Y-chromosome) fluorescence in situ hybridization (FISH) analysis was performed according to the manufacturer's protocol (Cambio, Cambridge, U.K., http://www.cambio.co.uk). Serial sections from paraffin-embedded whole pancreas block were cut (4-µm thickness) and prepared for procedure using tissue microarray (TMA). TMA permitted the combination of all tissue specimens, including positive and negative controls, on one slide. Tissue sections on coated slides were dewaxed, rehydrated, treated with sodium thiocyanate solution and pepsin, fixed, and dehydrated. After air drying, 15–20 µl of denatured nucleotide probe was added to each slide, covered with a coverslip, and sealed. After overnight hybridization at 37°C, the coverslip was removed, and serial washings were performed. Nuclei were counterstained with 4,6-diamidino-2-phenylindole. Slides were examined by fluorescent microscope (BX-41; Olympus, Tokyo, http://www.olympus-global.com) with an appropriate filter system.
Statistical Analysis
The Statistica software package (StatSoft, Tulsa, OK, http://www.statsoft.com) was used for statistical analysis. Results were analyzed by nonparametric Student's t test. Values of p < .05 were considered statistically significant.
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RESULTS |
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Two routes were used for ESC-derived HSCT: standard (i.v.) and intra-bone marrow injections. Nine of 10 mice from the IBM group and 5 of 8 from the i.v. group did not become hyperglycemic, in contrast to the control group, in which 8 of 9 mice were euthanized because of diabetes (Fig. 1A). Differences in the development of hyperglycemia were statistically significant between the IBM and the control groups (p < .01), between the i.v. and the control groups (p < .05), and between the IBM and the i.v. groups (p < .05). To exclude the influence of TBI on disease progression, four NOD mice were irradiated but injected with 0.9% NaCl. Two died 2 weeks after TBI, and the other two developed diabetes and were euthanized (Fig. 1A).
Twenty-four weeks after transplantation, the level of ESC-derived hematopoietic chimerism (H2-Kb+) in peripheral blood of IBM group was 9.1% ± 6.7% for CD45+ cells, 8.1% ± 4.3% for CD3+ cells, and 3.3% ±3.1% for CD19+ cells, whereas in the i.v. group, ESC-derived chimerism was 3.6% ± 2.7% for CD45+ cells, 2.1% ± 1.9% for CD3+ cells, and 1.6% ± 1.5% for CD19+ cells (Fig. 2A, 2C, 2D). The difference in total (CD45+) and T lymphocyte (CD3) chimerism between IBM and i.v. groups was statistically significant (p < .01). ESC-derived B lymphopoiesis was less than ESC-derived T lymphopoiesis and did not differ significantly between groups. There was no statistically significant difference in CD45+ chimerism noted at 10 and 24 weeks after ESC-derived HSCT. The end-point analysis of peripheral blood chimerism (40 weeks post-transplantation) revealed no changes compared with 24 weeks. ESC-derived chimerism in the spleen at 40 weeks post-ESC transplantation (ESCT) was slightly higher compared with peripheral blood, but the difference was not statistically significant (Fig. 2B). The flow cytometric analysis of splenocytes showed the presence of CD4+ and CD8+ cells derived from transplanted ESC-derived HSCT. The ESC-derived CD4/CD8 ratio was 0.3 ± 0.06 and did not significantly differ between groups or the level of total ESC-derived chimerism.
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was detected only in those cultures containing GAD65 and splenocytes from untreated NOD mice. In contrast, the level of IFN secreted by splenocytes derived from NOD mice transplanted with ESC-derived HSC cultured with the same concentration of GAD65 was comparable to that of the negative control (BALB/c splenocytes cultured with GAD65) (Fig. 4A).
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To investigate whether β islet cells originated from male donor ESC-derived HSC, we evaluated nuclei for the presence of Y-chromosome in pancreatic sections of six female NOD mice treated with ESCT. Approximately 500 nuclei in endocrine and exocrine parts of each pancreas were analyzed. FISH analysis of pancreatic sections demonstrated the absence of ESC-derived Y-chromosome-positive nuclei (0%) in chimeric animals. As a result of partial nuclear sampling in thin sections, FISH analysis yielded a Y-chromosome-positive nuclei undercount of approximately 80% in positive controls. No Y-chromosome signal was detected in either negative controls (female nontreated NOD mice) or experimental group samples (six normoglycemic NOD mice after ESCT) (data not shown).
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DISCUSSION |
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Full donor chimerism was not achieved in NOD mouse recipients of ESC-derived HSC. Compared with other strains such as BALB/c, NOD mice are somewhat resistant to allogeneic engraftment, especially when donor lymphocytes do not accompany the graft. These observations are consistent with previous reports demonstrating a relative resistance to allo-engraftment in NOD mice and the requirement for donor lymphocytes to achieve full donor chimerism [4, 16]. Surprisingly, full donor engraftment (that is, 100% donor hematolymphopoiesis) was not required to induce islet cell tolerance, and ESC-derived engraftment as low as 5% exerted a profound antidiabetic effect. This effect cannot be attributed to the conditioning regimen itself, as controls subjected to conditioning in the absence of ESC-derived HSCT died or developed diabetes. Since the prevention of diabetes did not require the complete replacement of host hematopoietic and immune systems, it appears that the reintroduction of even small numbers of diabetic-resistant hematopoietic stem cells is capable of favorably modulating impaired mechanisms of tolerance in autoimmune diseases.
We have confirmed our previous report on the superior engraftment of ESC-derived HSC from IBM injection over standard i.v. injection [10]. Fewer cells for IBM injection were required to obtain higher level of chimerism. The mechanism for higher engraftment from IBM injection is currently unknown, but possible mechanisms include impaired ESC-derived HSC selectin and/or integrin expression required for marrow homing following i.v. administration and/or differentiation of ESC-derived HSC into both hematopoietic and supportive and/or nurturing marrow mesenchymal cells (studies currently in progress).
Multiple studies suggest that both hematopoietic and mesenchymal bone marrow-derived stem cells have a greater than previously expected potential to "transdifferentiate" into diverse tissue types [17–20]. For example, Ianus et al. demonstrated that adult bone marrow-derived stem cells engrafted into the pancreata of mice; recipients expressed a β cell phenotype [19]. However, the phenomenon of the transdifferentiation remains elusive, and the theory of adult stem cell transdifferentiation has recently been called into question [21, 22]. To investigate whether β islet cells originated from male donor ESC-derived hematopoietic stem cells, we evaluated nuclei for the presence of Y-chromosome in pancreatic sections of female chimeric mice. We found no evidence to support plasticity or transdifferentiation of ESC-derived HSC into pancreatic islet cells. The absence of a Y-chromosome-positive signal in pancreatic islet nuclei from chimeric mice suggests that transdifferentiation of ESC-derived HSC into pancreatic cells did not occur and thus did not contribute to the prevention of diabetes in NOD chimeric mice.
In our study, donor chimerism was achieved by the introduction of stem cells lacking committed hematopoietic cells (e.g., mature T cells, B cells). This makes ESC-derived HSCT unique compared with peripheral blood or bone marrow-originated HSCT. Furthermore, nonspecific modulation of the host immune system from ESC-derived HSC did not occur since robust responses to third-party antigens ruled out nonspecific immune suppression. ESC-derived HSCT did, however, result in islet cell tolerance, as confirmed by glucose levels, survival, histology, and antigen-specific nonresponsiveness to GAD65, a diabetic-specific islet cell antigen.
The exact mechanism of ESC-derived HSC-induced tolerance to islet cells remains unclear. However, unlike malignancies, in which donor allogeneic lymphocytes transferred with the stem cell graft are responsible for both GVHD and GVL, in case of autoimmune diseases, transfer and establishment of mixed chimerism by using ESC-derived HSC to transplant only the hematopoietic progenitor cell compartment without contaminating lymphocytes conveys a potent tolerizing GVA effect without GVHD. Whether the mechanism of observed tolerance is through T regulatory cells, suppression or clonal deletion is unknown. Since lymphocytes including CD4+ and CD8+ cells matured in vivo from ESC-derived HSC, we speculate that the induction of tolerance, which occurred despite persistent host hematolymphopoiesis, may be due to ESC-derived immunoregulatory cells. Interestingly, Fandrich et al. [23] have reported that partial mixed chimerism following rat embryonic stem cell-like cells transplantation resulted in induction of tolerance. The same authors suggest that the mechanism of this tolerance is via clonal deletion of alloreactive T cells.
So far, there have been few reports in the literature of successful in vivo application of ESC or ESC-derived cells to treat and/or cure disease in an animal model. This study demonstrates a beneficial in vivo therapeutic application of ESC-derived HSC to induce islet cell tolerance and offers evidence that even in the absence of post-transplant immunosuppression, a tolerizing GVA effect may occur without GVHD. If confirmed in human studies, ESC-derived HSC may offer the hope of curing autoimmune diseases without the adverse and sometimes lethal consequences of GVHD currently associated with bone marrow or peripheral blood stem cell transplantation.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...References |
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REFERENCES |
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  R. K. Burt, Y. Loh, W. Pearce, N. Beohar, W. G. Barr, R. Craig, Y. Wen, J. A. Rapp, and J. Kessler Clinical Applications of Blood-Derived and Marrow-Derived Stem Cells for Nonmalignant Diseases JAMA, February 27, 2008; 299(8): 925 - 936. [Abstract] [Full Text] [PDF]
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