Hammerman Xenotransplantation of organ primordia Curr Opin Org TX 2014

REVIEW

CURRENTOPINION Classic and current opinion in embryonic organ

transplantation

1087-2418 � 2014 Wolters Kluwer

Marc R. Hammerman

Purpose of review

Here, we review the rationale for the use of organs from embryonic donors, antecedent investigations andrecent work from our own laboratory, exploring the utility for transplantation of embryonic kidney andpancreas as an organ replacement therapy.

Recent findings

Ultrastructurally precise kidneys differentiate in situ in rats following xenotransplantation in mesentery ofembryonic pig renal primordia. The developing organ attracts its blood supply from the host. Engraftmentof pig renal primordia requires host immune suppression. However, beta cells originating from embryonicpig pancreas obtained very early following initiation of organogenesis [embryonic day 28 (E28)] engraftlong term in nonimmune-suppressed diabetic rats or rhesus macaques. Engraftment of morphologicallysimilar cells originating from adult porcine islets of Langerhans occurs in animals previously transplantedwith E28 pig pancreatic primordia.

Summary

Organ primordia engraft, attract a host vasculature and differentiate following transplantation to ectopicsites. Attempts have been made to exploit these characteristics to achieve clinically relevant endpoints forend-stage renal disease and diabetes mellitus using animal models. We and others have focused on use ofthe embryonic pig as a donor.

Keywords

chronic renal failure, diabetes mellitus, organ primordia, organogenesis, xenotransplantation

Departments of Medicine, and Cell Biology and Physiology,WashingtonUniversity School of Medicine, St. Louis, Missouri, USA

Correspondence toMarc R. Hammerman, MD, Renal Division, Box 8126,Department of Medicine,Washington University School of Medicine, 660S. Euclid Ave. St. Louis, MO 63110, USA. Tel: +1 314 362 8233; fax: +1314 362 8237; e-mail: [email protected]

Curr Opin Organ Transplant 2014, 19:133–139

DOI:10.1097/MOT.0000000000000054

INTRODUCTION

It has been known for close to a hundred yearsthat primordia of mammalian organs, oncemorphologically defined, can maintain themselvesand undergo differentiation following transplan-tation to sites such as the mesentery, kidney capsuleor anterior eye chamber [1]. Classically, transplan-tation to mesentery was deemed to be particularfavorable in terms of its permitting undisturbedexpansion of a growing organ primordium, hencemorphogenesis that is not physically constrainedover time, and resulting in vascularization by hostblood vessels [2]. Under some circumstancesengraftment was shown to occur following trans-plantation of embryonic organs to adult animals ofa different species without the need to immunesuppress hosts [3]. Within the past few decades,efforts have been made by us and others to exploitthe body of classic knowledge about embryonicorgan transplantation to achieve a therapeuticend. Important studies antecedent to our owninclude those of Woolf et al. [4] who explored the

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possibility of adding new nephrons to the mamma-lian kidney via isotransplantation of embryonicmetanephric tissue within renal parenchyma andreported that functioning nephrons can be added tomammalian kidneys by this technique in neonatalmice; those originating in the Lazarow [5] andBrown [6] laboratories showing that experimentaldiabetes can be controlled in rats by isotransplanta-tion of embryonic pancreas and that a novel organconsisting of islets of Langerhans in stroma withoutexocrine tissue differentiates in hosts postproce-dure; and the work of Eloy et al. [7] who demon-strated that chick embryo pancreatic transplants

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KEY POINTS

� Organ primordia engrafts attract a host vasculature anddifferentiate following transplantation to ectopic sites.

� Attempts have been made to exploit thesecharacteristics to achieve clinically relevant endpointsfor end-stage renal disease and diabetes mellitus usinganimal models.

� We and others have focused on the use of theembryonic pig as a donor.

� What happens after transplantation of embryonickidneys or pancreas as defined by what sort ofstructure differentiates, whether it engrafts at theimplantation site or migrates elsewhere, and bywhether or not host immune suppression is required forengraftment, varies from experimental case to case.

� It is impossible to know what will happen after anembryonic organ obtained at a given developmentalstage is isotransplanted, allotransplanted ortransplanted across a narrow or wide xenogeneicbarrier until one does the experiment.

Organogenesis and organ regeneration and repair after transplantation

reverse experimental diabetes in rats without a hostimmune-suppression requirement. Although thetransplantation of human embryonic organs inhuman hosts has been contemplated by others,we have focused on the use of embryonic organsfrom the pig, a physiologically suitable donor forhuman pancreas or kidney replacement [reviewed in[8]].

TRANSPLANTATION OF EMBRYONICORGAN PRIMORDIA TO REPLACEFUNCTION OF FAILED ORGANS

We have shown that it is possible to ‘grow’ newkidneys [9–11] or endocrine pancreatic tissue[11–17] in situ via xenotransplantation of organprimordia from pig embryos (organogenesis of theendocrine pancreas or kidney). The developingrenal organ attracts its blood supply from the host[10]. In the case of pancreas, selective developmentof endocrine tissue takes place posttransplantation,developing beta cells enter lymphatic vessels andengraft in mesenteric lymph nodes from which theysecrete insulin in response to elevated blood glucose[11–17]. Glucose intolerance can be corrected informerly diabetic rats [11–14,16] and amelioratedin rhesus macaques [15,17] on the basis of porcineinsulin secreted in a glucose-dependent manner bybeta cells originating from transplants. In the caseof kidney, an anatomically correct functionalorgan differentiates in situ at the transplantationsite [9–11]. Life can be prolonged in an otherwise

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anephric rat on the basis of renal function providedby a single transplanted rat renal primordium, theureter of which is anastomosed to a ureter of thehost [18]. If obtained within a ‘window’ early duringembryonic pancreas development, pig pancreaticprimordia engraft in nonimmune-suppresseddiabetic rats [11–14,16] or rhesus macaques[15,17]. In contrast, engraftment of pig renalprimordia transplanted into rats requires hostimmune suppression [11].

Shown in Fig. 1a is a photograph of an E28 pigrenal primordium. The ureteric bud is labeled (ub).Figure 1b is a photograph of a kidney that hasdeveloped in situ. The ureter is labeled (u).Figure 1c is a photomicrograph that shows an E28pig renal primordium. It consists of undifferentiatedstroma, branched ureteric bud and primitivedeveloping nephrons (arrow) (11). The renal cortexof the kidney shown in Fig. 1b consists of normal-appearing glomeruli (g), proximal tubules (pt) anddistal tubules (dt) (Fig. 1d). Its glomeruli arevascularized by host vessels that stain (brown) withantirat endothelial antigen 1 (RECA-1), which isspecific for rat endothelium (Fig. 1e).

An E28 pig pancreatic primordium with separatedorsal pancreas (dp) and ventral pancreas (vp)components is shown as an inset in Fig. 2a.Figure 2b shows the mesentery of a streptozotocin(STZ) diabetic rhesus macaque at the time oftransplantation. A primordium between sheetsof mesentery is delineated (arrowhead). Figure 3shows photomicrographs originating from amesenteric lymph node of a rhesus macaque trans-planted previously with E28 pig pancreatic primor-dia in mesentery. Sections in Fig. 3a and c are stainedwith an antiinsulin antibody. Sections in Fig. 3b andd are incubated with control serum. Individual cellsthat stain positive (red) are present in medullarysinus (arrow). The cells are polygonal, consistentwith a beta cell identity (Fig. 3c arrow). Nopositive-staining cells are found in sections incu-bated with control serum (Fig. 3b and d). Engraft-ment of pig tissue in the mesenteric lymph nodesis documented using in-situ hybridization for por-cine proinsulin mRNA. Cells expressing porcinemRNA stain with use of an antisense probe toporcine proinsulin mRNA (Fig. 3e), but not a senseprobe (Fig. 3f).

As noted, glucose tolerance can be nearly nor-malized in nonimmune-suppressed diabetic rhesusmacaques following transplantation of E28 pigpancreatic primordia [15,17]. Exogenous insulinrequirements are reduced in transplanted macaques,and animals have been weaned off insulin for shortperiods of time, but not permanently as is the casefor rats transplanted with pig pancreatic primordia

Volume 19 � Number 2 � April 2014

IGURE 1. Photographs (a and b) and photomicrographs–e) of: (a) a renal primordium freshly dissected from an E28ig embryo; (b) a developed pig renal primordium 7 weeksllowing transplantation after removal from mesentery; (c) anal primordium freshly dissected from an E28 pig embryo.ranched ureteric bud and developing nephron (arrow);) cortex of a developed pig renal primordium in ratesentery 7 weeks following transplantation. Glomerularapillary loop (arrow); (e) a glomerulus within the cortex ofdeveloped pig renal primordium in rat mesentery 7 weeksllowing transplantation stained with rat endothelial cellntigen 1 (RECA-1). ub, ureteric bud; u ureter; pt, proximalbule; dt, distal tubule; g, glomerulus. Scale bar 80 mm (a, c);mm (b); 10 mm (d); 5 mm (e). Reproduced with permission0,11].

FIGURE 2. (a) Photograph of a pancreatic primordiumfreshly dissected from an E28 pig embryo. (b) A pancreaticprimordium implanted between sheets of mesentery in arhesus macaque. dp, dorsal pancreas; vp, ventral pancreas.Scale bar 10 mm (a). Reproduced with permission [15].

Opinion in embryonic organ transplantation Hammerman

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[11–14,16]. The most likely explanation for thedifference between rats and macaques is thatmacaques weigh 20 times as much as rats. AnSTZ-diabetic rat can be rendered normoglycemiclifelong with no exogenous insulin requirementby transplantation of 5–8 pig pancreatic primordia.Extrapolating, it would take 100–160 primordia torender a diabetic rhesus macaque independent ofexogenous insulin. This would require the sacrificeof about 7–12 pregnant sows and multiple surgerieswith the attendant complications.

In lieu of increasing the numbers of trans-planted primordia or transplant surgeries in diabeticrhesus macaques, we embarked on a series ofexperiments to determine whether porcine islets,a more easily obtainable and possibly more robustsource of insulin-producing cells, could be substi-tuted for animals in which embryonic pig pancreasalready had engrafted. To this end, we implantedadult porcine islets beneath the capsule of onekidney from rats [16] or macaques [17] that severalweeks earlier had been transplanted with E28pig pancreatic primordia in mesentery. Weemployed the renal subcapsular site for isletimplantation so that we could differentiateengrafted porcine tissue originating from the isletsfrom tissue originating from prior mesenteric E28pig pancreatic transplants that never engraft in hostkidney. In this setting, the contralateral (nontrans-planted) kidney served as a control as did kidneysfrom rats or macaques implanted with isletswithout prior transplantation of E28 pig pancreaticprimordia in mesentery. Figure 4 shows sections

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FIGURE 3. Photomicrographs of mesenteric lymph nodes from a STZ-diabetic diabetic rhesus macaque posttransplantation ofE28 pig pancreatic primordia. Sections (a) and (c) are stained with an anti-insulin antibody. Sections (b) and (d) are stainedusing a control serum. Arrow delineates medullary sinus (a). Arrow delineates polygonal cell (c). In-situ hybridization wasperformed using antisense (e) or sense probes (f). Arrows delineate a cells in consecutive sections to which the antisense probebinds (e). Scale bars 80 mm (a and b); 10 mm (c–e). Reproduced with permission [15].


from a kidney of a STZ-diabetic rat (Fig. 4a and b) orrhesus macaque (Fig. 4c and d) implanted withporcine islets following transplantation of E28 pigpancreatic primordia in mesentery. Sections arestained using antiinsulin antibodies (Fig. 4a and c)or control serum (Fig. 4b and d). Cells that stain forinsulin (Fig. 4a and c), but not with control serum(Fig. 4b and d) are present in an expanded renalsubcapsular space. Nuclei of cells in the subcapsularspace hybridize to antisense robes for porcine


proinsulin mRNA, but not sense probes [16,17].Neither cells that stain for insulin nor cells to whichthe probe for porcine proinsulin mRNA binds arepresent in contralateral (nonimplanted) kidneys ofSTZ diabetic rats or macaques in which E28 pigpancreatic primordia were transplanted previouslyin mesentery or in kidneys from STZ diabetic rats ormacaques into which porcine islets are implantedwithout prior transplantation of E28 pig pancreaticprimordia in mesentery [16,17].


FIGURE 4. Sections of the islet-implanted kidney from a STZ-diabetic Lewis rat (a and b) or rhesus macaque (c and d)transplanted with E28 pig pancreatic primordia in mesentery followed by porcine islets in the renal subcapsular space stainedusing antiinsulin antibodies (a and c) or control antiserum (b and d). PT, proximal tubule. RC, renal capsule. Arrows, positivelystaining cells (a and c); negatively staining cells (b and d). Scale bar 10 mm. Reproduced with permission [16,17].


To ascertain whether cells originating from kid-ney-implanted porcine islets function in rats orrhesus macaques, we determined whether the glu-cose tolerance of STZ-diabetic animals normalizedpartially by prior transplantation of E28 pig pancre-atic primordia in mesentery was rendered normal bysubsequent islet implantation, and measured glu-cose-stimulated insulin release from islet implantedkidneys in vitro. Glucose tolerance tests in rats werenormalized by subsequent implantation of porcineislets in one kidney [16]. The glucose tolerance ofmacaques normalized partially by prior transplan-tation of E28 pig pancreatic primordia in mesenterywas not improved by subsequent implantation ofislets in kidney. However, a rapid release of insulinby macaque kidney slices was demonstrated in vitroin response to elevation of glucose levels across thethreshold for insulin release [17].

Intact porcine islets do not engraft followingrenal subcapsular implantation [16,17]. Rather, apopulation of cells originating from donor isletswith beta cell morphology that express insulin


and porcine proinsulin mRNA engraft in kidneysof rats transplanted previously with E28 pig embry-onic pancreas. Our observations are consistent withthe induction of tolerance to a cell component ofadult porcine islets by previous transplantation ofE28 pig pancreatic primordia in rats. We designatethe phenomenon organogenetic tolerance [19].Whatever its cause might be, induction of organo-genetic tolerance to porcine islets in humans withdiabetes mellitus would enable the use of pigs asislet donors with no host immune suppressionrequirement.

Ours is not the only group that has undertakenstudies to ascertain whether transplantation embry-onic pig renal or pancreatic primordia can beexploited to treat renal failure or diabetes mellitusin humans. Detailed comparisons of the findingsdescribed herein and the investigations of othersare published elsewhere [8,20,21,22

&

] and will notbe repeated here. Our body of work includes notonly studies for which embryonic pigs serve asdonors for organs transplanted in mesentery, but



also investigations in which organs from embryonicrats were transplanted in mesentery and elsewhere.The findings are broadly confirmatory of precedinginvestigations. Thus, we recapitulated the findingsof Woolf et al. [4] that nephrons can be added todeveloped kidneys via transplantation of renal pri-mordia beneath the capsule [23] prior to introduc-ing use of the mesenteric site [9–11,18,23,24].Interestingly, allotransplantation of renal primordia(rat to rat) can be performed in mesentery withouthost immune suppression [24]. We confirmed theobservations of Hegre et al. [5] and Brown et al. [6], inthat the novel organ they described (islets of Lan-gerhans within connective tissue stroma) differen-tiates post isotransplantation in rat [25]. Weextended [11–17] the observation of Eloy et al. [7]that engraftment following xenotransplantation ofembryonic pancreas can occur in nonimmune sup-pressed hosts (chick-to-rat) to different xenogeneicpairs (pig-to-rat and pig-to-rhesus macaque [11–17].

However, broadly confirmatory our findingsmay have been of classic observations [1–7], thereis an element of unpredictability to them. Forexample, the results of transplanting embryonickidney to mesentery (formation of a structurallycorrect renal organ) are different from results oftransplanting embryonic pancreas (formation ofthe novel organ described above or disseminationof beta cells along a lymphatic distribution).Furthermore, outcomes following transplantationof embryonic pancreas differ not only dependingon whether isotransplantation is carried out (novelorgan), but also depending on what xenogeneicbarrier is crossed (rat-to-mouse transplantationresults in formation of the novel organ and requireshost immune suppression; pig-to-rat or pig-to-rhe-sus macaque results in lymphatic dissemination ofbeta cells and no immune suppressions is required).In this regard, it has been impossible to predictwhat sort of structure will differentiate, or whetherit will engraft at the implantation site or migrateelsewhere. Furthermore, it has been impossibleto know whether or not host immune suppressionif applicable (for xenotransplantation) will berequired for engraftment.

SUMMARY

A major advantage inherent in the use of embryonickidney or pancreas for transplantation relative tomore pluripotent undifferentiated cells is that theformer differentiate spontaneously along definedorgan-committed lines, albeit with a different out-come relative to what would occur if the primordiaremained undisturbed within the embryo. In thecase of pig renal primordia transplanted in


mesentery, a kidney differentiates in situ with hostvasculature [9–11]. In the case of embryonic pigpancreas all that remains in hosts posttransplanta-tion are beta cells engrafted in mesenteric lymphnodes, for which glucose sensing and insulin-releas-ing mechanisms are functionally linked [11–17].Transplantation of embryonic pancreas is one ofmany cellular strategies that can be employed toreplace beta cell function. Others include isletimplantation and transplantation of stem cells thatdifferentiate into insulin producers [8,21,22

&

]. Incontrast, applications for cell transplantation toreplace the function of a structurally complex organsuch as the kidney are more limited. In order forglomerular filtration, reabsorption, and secretion offluid and electrolytes to take place in a manner thatwill sustain life; individual nephrons must be inte-grated in three dimensions with one another andwith a collecting system, the origin of which is yetanother separate structure, the ureteric bud. Con-comitantly, vascularization must occur in a uniqueorgan-specific manner from endothelial precursorsthat may originate from both inside and outside ofthe developing renal primordium. Although it isconceivable that endocrine functions of the kidney,such as erythropoietin production, could bereplaced by transplanting one particular type ofrenal cell, recapitulation of glomerular filtrationand reabsorption in kidneys, and excretion of urinewill be a much more formidable challenge for renalcell therapy.

Acknowledgements

The author acknowledges lectures by the late Dr ViktorHamburger at Washington University in 1966–1967delivered to him as part of his undergraduate ComparativeAnatomy and Embryology course which, at least in retro-spect, were inspirational. Juvenile Diabetes ResearchFoundation 1–2008–37; National Institutes of HealthP30 DK079333; Washington University Selina ConnerMemorial Research Fund and Endowment.

Conflicts of interest

There are no conflicts of interest.

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3. Greene HSN. Attributes of embryonic tissues after growth and developmentin heterologous hosts. Cancer Res 1955; 15:170–172.



4. Woolf AS, Palmer SJ, Snow ML, Fine LG. Creation of a functioning chimericmammalian kidney. Kidney Int 1990; 38:991–997.

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6. Brown J, Molnar JG, Clark W, Mullen Y. Control of experimental diabetesmellitus in rats by transplantation of fetal pancreases. Science 1974;184:1377–1379.

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9. Rogers SA, Talcott M, Hammerman MR. Transplantation of pig renal anlagen.ASAIO J 2003; 49:48–52.

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11. Rogers SA, Liapis H, Hammerman MR. Normalization of glucose posttrans-plantation of pig pancreatic anlagen into nonimmunosuppressed diabetic ratsdepends on obtaining anlagen prior to embryonic day 35. Transplant Immunol2005; 14:67–75.

12. Rogers SA, Chen F, Talcott M, Hammerman MR. Islet cell engraftment andcontrol of diabetes in rats following transplantation of pig pancreatic anlagen.Am J Physiol 2004; 286:E502–E509.

13. Rogers SA, Chen F, Talcott M, et al. Glucose tolerance normalizationfollowing transplantation of pig pancreatic primordia into nonimmunosup-pressed diabetic ZDF rats. Transplant Immunol 2006; 16:176–184.

14. Rogers SA, Hammerman MR. Normalization of glucose posttransplantationinto diabetic rats of pig pancreatic primordia preserved in vitro. Organogen-esis 2008; 4:48–51.


15. Rogers SA, Chen F, Talcott MR, et al. Long-term engraftment followingtransplantation of pig pancreatic primordia into nonimmunosuppressed dia-betic rhesus macaques. Xenotransplantation 2007; 14:591–602.

16. Rogers SA, Mohanakumar T, Liapis H, Hammerman MR. Engraftment of cellsfrom porcine islets of Langerhans and normalization of glucose tolerancefollowing transplantation of pig pancreatic primordia in nonimmune sup-pressed diabetic rats. Am J Pathol 2010; 177:854–864.

17. Rogers SA, Tripathi P, Mohanakumar T, et al. Engraftment of cells fromporcine islets of Langerhans following transplantation of pig pancreaticprimordia in nonimmune suppressed diabetic rhesus macaques. Organogen-esis 2011; 7:154–162.

18. Rogers SA, Hammerman MR. Prolongation of life in anephric rats following denovo renal organogenesis. Organogenesis 2004; 1:22–25.

19. Hammerman MR. Organogenetic tolerance. Organogenesis 2010; 6:270–275.

20. Hammerman MR. Transplantation of renal primordia: renal organogenesis.Pediatr Nephrol 2007; 22:1991–1998.

21. Hammerman MR. Xenotransplantation of pancreatic and kidney primordia:where do we stand? Transplant Immunol 2009; 21:93–100.

22.&

Hammerman MR. Xenotransplantation of embryonic pancreas for treatmentof diabetes mellitus in nonhuman primates. J Biomed Sci Eng 2013; 6:6–11.

Review of a body of work directed toward the development of a novel xenotrans-plantation therapy for diabetes mellitus.23. Rogers SA, Lowell JA, Hammerman NA, Hammerman MR. Transplantation of

developing metanephroi into adult rats. Kidney Int 1998; 54:27–37.24. Rogers SA, Liapis H, Hammerman MR. Transplantation of metanephroi across

the major histocompatibility complex in rats. Am J Physiol 2001; 280:R132–R136.

25. Rogers SA, Liapis H, Hammerman MR. Intraperitoneal transplantation ofpancreatic anlagen. ASAIO J 2003; 49:527–532.


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