Functional Human to Mouse Adipose Tissue Xenotransplantation
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41 Functional human to mouse adipose tissue xenotransplantation Vitaly Ablamunits, Simon Klebanov1, Sharon Y Giese2 and Kevan C Herold Department of Immunobiology, Yale University School of Medicine, 300 George Street, Suite 334-D, New Haven, Connecticut, USA 1Obesity Research Center, St. Luke’s Hospital, New York, New York, USA 2Lenox Hill Hospital and Private Ambulatory Clinic, New York, New York, USA (Correspondence should be addressed to V Ablamunits; Email: [email protected]) Abstract White adipose tissue (WAT) produces a number of metabo- human leptin in circulation, reduced body mass gain, and lically important factors and, therefore, some inborn errors of amelioration of hepatic steatosis. Food consumption and metabolism may potentially be corrected by transplantation of plasma insulin levels were reduced only in recipients of 5 ml normal allogeneic WAT.Toexplore the ability of human WAT WAT. Transfer of 2!107 human mononuclear cells to reject (HuWAT) to compensate for a missing factor and to induce WAT as an allograft was ineffective and resulted only in some allogeneic immune response, we created leptin-deficient, reduction of circulating leptin and a limited damage to the immunodeficient mice and transplanted them with either 2.5 WAT grafts followed by the loss of human leukocytes. or 5 ml HuWAT. Recipient mice showed stable levels of Journal of Endocrinology (2012) 212, 41–47 Introduction and how graft rejection can be avoided. We have reported that mouse WAT grafts transplanted into leptin-deficient mice A growing evidence indicates that white adipose tissue may be monitored by reversal of the obese phenotype (WAT) is an important endocrine organ secreting a number (Klebanov et al. 2005) and that WAT allografts are acutely of factors collectively known as adipokines. Some of these rejected unless the host is immunosuppressed (Ablamunits factors, such as leptin (Zhang et al. 1994) and adiponectin et al. 2007). To study human responses to WAT allograft, we (Scherer et al. 1995), are unique for adipocytes, whereas decided to generate a humanized mouse that carries others, such as visfatin, apolipoprotein E (ApoE), lipoprotein functional human WAT (HuWAT). For that, we bred lipase (LPL), plasminogen activator inhibitor 1 (PAI1), and leptin-deficient (ob/ob), immunodeficient (Rag1 knockout) insulin-like growth factor 1 (IGF1), are also produced by mice. Our hypothesis was that HuWAT xenograft might also other cell types (Doglio et al. 1987, Sawdey & Loskutoff 1991, be functionally monitored by the reversal of obese phenotype, Zechner et al. 1991, Fukuhara et al. 2005). In humans, whereas a transfer of irrelevant human peripheral blood congenital isolated leptin deficiency is responsible for severe mononuclear cells (PBMC) would initiate allogeneic immune early onset obesity (Montague et al. 1997, Strobel et al. 1998), response to the HuWAT transplant and destroy it with while secondary leptin deficiency due to the paucity of WAT subsequent recurrence of leptin deficiency. Here, we report is associated with lipodystrophic diabetes (Gold & Steinbach metabolic and morphologic evidence of long-term survival 1967, Seip & Trygstad 1996, Pardini et al. 1998). Deficiencies and function of HuWAT in the mouse and our attempt to of other factors have been reported in humans, such as reject it as an allograft. GH-resistant dwarfism due to IGF1 deficiency (Woods et al. 1996, Bonapace et al. 2003), hyperlipoproteinemia caused by mutated ApoE or LPL genes (Zannis & Breslow 1980, Materials and Methods Ghiselli et al. 1981, Hayden & Ma 1992), and a bleeding disorder due to PAI1 deficiency (Dieval et al. 1991). These Animals conditions may, in principle, be treated by WAT trans- plantation (WATTx) for replacement of a missing factor. All mouse experiments were approved by the Yale Insti- Two major approaches may be suggested for WATTx: tutional Animal Care and Use Committee. Leptin-deficient genetic modification of autologous WAT, including WAT (B6.V-Lepob/J), Rag1-deficient (B6.129S7-Rag1tm1Mom), and differentiated from WAT-derived stem cells (Zuk et al. 2001), regular C57BL/6J (B6) mice were purchased from The and allogeneic WATTx from normal human donor. In both Jackson Laboratory (Bar Harbor, ME, USA). They were scenarios, however, it would be important to know whether housed in specific pathogen-free environment and used for the graft is recognized by the immune system of the recipient breeding or as donors of WAT. Journal of Endocrinology (2012) 212, 41–47 DOI: 10.1530/JOE-11-0201 0022–0795/12/0212–041 q 2012 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/02/2021 06:13:18PM via free access 42 V ABLAMUNITS and others . Human fat secretes leptin in the mouse Generation of immunodeficient leptin-deficient mice Diagnostics, Deerfield Beach, FL, USA). For insulin C tolerance test (ITT), mice were fasted for 3 h, injected with Rag1-deficient females were mated with B6.V-Lepob/ Humulin R (Eli Lilly and Company), i.p. (2 U/kg body heterozygote males. Resulting heterozygotes for both K K C mass), and blood glucose was measured before and 15, 30, and mutations were intercrossed to produce Rag1 / Lepob/ 60 min after the injection. mice, which were identified as IgG negative by ELISA and C V-Lepob/ by PCR (primers: forward, gctatggctggttcttcac; reverse, atccaggctctctggcttc) followed by a restriction enzyme Histology digestion (Hirasawa et al. 1997). These mice were intercrossed K/K ob/ob Upon termination of experiments, HuWAT grafts, pancreas, and double mutants (Rag1 , V-Lep ) designated here as and liver of mice were fixed in 4% formal saline, paraffin Rag/ob were identified by the obese phenotype. To render embedded, and stained with hematoxylin and eosin (H&E). these mice fertile, they were subsequently transplanted with Morphology was studied using Axioplan 2 microscope (Carl 3–4 ml gonadal WAT from B6 donors and caged as breeding K/K ob/ob Zeiss Microimaging, Inc., Thornwood, NY, USA). The pairs. Progeny of these breeders were all Rag1 , V-Lep degree of hepatic steatosis was evaluated semi-quantitatively and served as recipients for HuWAT. by scoring as 0 (no steatosis), 1 (patchy, !50% area), or 2 (severe). Islet size was measured by morphometry using HuWAT transplantation Axiovision 4.8.1 Software (Carl Zeiss). Subcutaneous HuWAT was obtained by liposuction from undisclosed donors who underwent this procedure for Flow cytometry cosmetic purposes. Each experiment represents a single Human leukocytes in circulation were analyzed by flow HuWAT donor so that each mouse within that experiment cytometry (FACSCalibur, BD Immunocytometry Systems, received equal amount of WATof the same origin and quality. San Jose, CA, USA) using mouse anti-human CD45, CD4, Samples were irradiated (1000 Rad, X-RAD 320 irradiator, and CD8 antibodies (BD Biosciences, San Diego, CA, USA). Precision X-Ray, North Branford, CT, USA) to prevent graft . Analysis of the data was performed using FlowJo Software vs host disease, and HuWAT (2 5 or 5 ml/mouse) was s.c. (TreeStar, Inc., Ashland, OR, USA). injected into multiple points of the dorsal area of recipient mice under isoflurane anesthesia (Forane; Baxter, Deerfield, IL, USA). Data analysis Data are expressed as meanGS.E.M. To assess the effectiveness HuWAT graft rejection of HuWATTx, statistical analysis was performed using GraphPad Prizm Version 5 Software (GraphPad Software, Normal human leukocytes were obtained from the Central Inc., San Diego, CA, USA). One-way ANOVA was applied Laboratory Facility, New York Blood Center (Long Island for comparison between the groups. Student’s two-tailed City, NY, USA), and PBMC were separated using Ficoll- unpaired or paired t-test was applied where applicable and Paque PLUS (GE Healthcare, Uppsala, Sweden) gradient P!0.05 considered significant. centrifugation. To reject allogeneic HuWAT, recipient mice were injected with 2!107 PBMC, i.p., 50 days after HuWATTx, and followed for additional 50 days before killing. Results Comparison of Rag/ob and regular ob/ob mice Measurements Rag/ob mice are leptin deficient and hence are overweight, Body weight was measured weekly. For food consumption, hyperphagic, hyperinsulinemic, and insulin resistant. mice were individually caged and food amount in the cage Figure 1A shows that Rag/ob mice are slightly less overweight hopper was measured daily for 5 days. To measure plasma than regular ob/ob mice with mean body mass at 3 months of leptin and insulin levels, blood samples were taken from the age: 59.0G0.9(nZ13) vs 64.1G1.1g (nZ9) respectively retro-orbital sinus of non-fasting mice on days 50 and 100 (PZ0.009). Despite this, their average food consumption after HuWATTx. Quantikine Human Leptin ELISA kit measured at 4 months of age was not different (5.3G0.32 vs (R&D Systems, Minneapolis, MN, USA) that detects no 5.6G0.32 g/day per mouse respectively; nZ3; not signi- more than 0.03 ng/ml of a cross-reacting substance in ob/ob ficant (NS); data not shown). Both, Rag/ob and ob/ob mice mice was used. Ultra Sensitive Mouse Insulin ELISA kit had similarly elevated non-fasting insulin levels of 21.9G1.5 (Crystal Chem, Inc., Downers Grove, IL, USA) was used for (nZ10) and 19.9G0.9ng/ml (nZ6) respectively (NS; measuring insulin in the same samples. Non-fasting glucose Fig. 1B). Non-fasting blood glucose levels at 3 months were was measured from the blood samples of the mice from the tip not significantly different (320G41 vs 255G22.8 mg/dl, NS; of the tail and using Easy Check glucose meter (Home Aide Fig. 1C). ITT using 2 U/kg body mass of Humalog revealed Journal of Endocrinology (2012) 212, 41–47 www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/02/2021 06:13:18PM via free access Human fat secretes leptin in the mouse . V ABLAMUNITS and others 43 AB transplantation, blood samples were taken from the mice for 25 75 * determination of human leptin in circulation.