Elutriated Stem Cells Derived from the Adult Bone MarrowDifferentiate into Insulin-Producing Cells In Vivo

and Reverse Chemical Diabetes

Svetlana Iskovich,1 Nitza Goldenberg-Cohen,2 Jerry Stein,3 Isaac Yaniv,3

Ina Fabian,4 and Nadir Askenasy1

An ongoing debate surrounds the existence of stem cells in the adult endowed with capacity to differentiate intomultiple lineages. We examined the possibility that adult bone marrow cells participate in recovery fromchemical diabetes through neogenesis of insulin-producing cells. Small-sized cells negative for lineage markersderived by counterflow centrifugal elutriation from the bone marrow were transplanted into mice made diabeticwith streptozotocin and sublethal irradiation. These cells homed efficiently to the injured islets and contributedto tissue revascularization. Islet-homed CD45-negative donor cells identified by sex chromosomes down-regulated GFP, expressed PDX-1 and proinsulin, and converted the hormone precursor to insulin. An estimated7.6% contribution of newly formed insulin-producing cells to islet cellularity increased serum insulin and sta-bilized glycemic control starting at 5 weeks post-transplant and persisting for 20 weeks. Newly differentiatedcells displayed normal diploid genotype and there was no evidence of fusion between the grafted stem cellsor their myeloid progeny and injured b-cells. Considering the extensive functional incorporation of insulin-producing donor cells in the injured islets, we conclude that the adult bone marrow contains a subset of smallcells endowed with plastic developmental capacity.

Introduction

Terminally differentiated organs are not only de-graded in adult life, but continuous replenishment of cells

maintains tissue mass and function. For example, the con-tinuous turnover of b-cells in adult life [1] is attributed both toproliferation of mature b-cells and activation of tissue pro-genitors [2–6]. In parallel to characterization of the slowprocess of physiological cell renewal from tissue progenitors,efforts are directed to design ways to foster organogenesis fortherapeutic purposes [7,8]. A preferred source of candidatestem cells for tissue regeneration is the bone marrow, one ofthe best-characterized developmental systems and an acces-sible source of primitive progenitors. The bone marrow isconstitutively connected to peripheral circulation, which isthe plausible route of dissemination of multipotent units ofrepair to all tissues.

In type 1 diabetes it is essential to abrogate the autoim-mune reaction that threatens any cell type with phenotypiccharacteristics (antigenic targets) of b-cells before attempts toregenerate the tissue [9]. Interventions aiming to reconstitutethe islet mass have yielded controversial data so far. It is

generally recognized that hematopoietic and mesenchymalstromal cells derived from the bone marrow and umbilicalcord blood (UCB) exert a positive effect on islet recoveryfrom chemical injury [10–26]. The therapeutic effects havebeen attributed primarily to facilitation of b-cell recoveryfrom chemical injury through support of b-cell viability andfunction [11,13,17,24], local immunomodulation [10,14,20,25], neovascularization [11,16,20], and local secretion ofgrowth factors [18,22]. Insulin production has been sporad-ically observed in donor cells [10,11,19,26]; however,significant conversion of adult cells from hematopoieticcompartments to produce insulin that would be of thera-peutic value has been observed only in transplants of bonemarrow cells (BMC) after myeloablative irradiation [12] andof UCB cells in neonate immunodeficient mice [27]. Datareported by these in vivo studies differ from the effectiveinduction of insulin production in vitro, which was con-vincingly demonstrated for both adherent [28] and non-adherent fractions of BMC [29] and UCB cells [30,31]. If thecapacity of differentiation is present in various cell types,negative reports might originate from suboptimal timing,detection technique, or selection of the subset of primitive

1Frankel Laboratory and 2Krieger Laboratory, Center for Stem Cell Research, and 3Department of Pediatric Hematology-Oncology,Schneider Children’s Medical Center of Israel, Petach Tikva, Israel.

4Department of Cell Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

STEM CELLS AND DEVELOPMENT

Volume 21, Number 1, 2012

� Mary Ann Liebert, Inc.

DOI: 10.1089/scd.2011.0057

86

cells derived from the bone marrow [32]. Progenitors derivedfrom this compartment undertake a default and dominanthematopoietic differentiation trait under most experimentalconditions [33].

One way to circumvent the predisposition of bonemarrow-derived progenitors to reconstitute the immuno-hematopoietic system during acute stages of islet injury is theuse of subsets that lack short-term hematopoietic activity[34,35]. The smallest elutriated fraction of the bone marrowhas poor radioprotective capacity, but elaborates delayedand durable long-term reconstituting potential [34–36]. Thissubset is endowed with the capacity to differentiate intomultiple non-hematopoietic lineages [37], including liver[38], and single cells contribute widely to reconstitution ofepithelial tissues [39]. In this study we examined the contri-bution of adult bone marrow-derived primitive cells torecovery from chemical diabetes through neogenesis ofinsulin-producing cells. Using an experimental approachdesigned according to our interpretation of the literature [9]and careful evaluation of the target tissue [32], we found thatadult bone marrow-derived cells home efficiently to theinjured islets, differentiate to produce insulin, and restorenear-euglycemic levels through contribution of *8% toinsulin-producing cells.

Materials and Methods

Animal preparation, diabetes, and transplantation

Mice used in this study were C57Bl/6J (B6, H2Kb,CD45.2), B6.SJL-Ptprca Pepcb/BoyJ (H2Kb, CD45.1), andC57BL/6-TgN(ACTbEGFP)1Osb (GFP, H2kb, CD45.2) pur-chased from Jackson Laboratories and housed in a barrierfacility. All procedures were approved by the InstitutionalAnimal Care Committee. Diabetes was induced in femalemice (aged 6–8 weeks) by 5 daily consecutive intraperitonealinjections of 60mg/g Streptozotocin (STZ; Calbiochem) [32].STZ was diluted in phosphate-buffered saline (PBS; BeitHaemek) and was used within 15 min of preparation. Bloodglucose levels were monitored with a standard glucometer(Accu-Chek Sensor; Roche Diagnostics) in mice fed ad libi-tum at constant daytime hours (9–11 AM). Diabetes wasconsidered at glucose levels exceeding 250 mg/dL in 2 con-secutive measurements. Glucose tolerance test was per-formed by intraperitoneal injection of 2 g glucose and bloodlevels measurements after 60 and 120 min. Before cell trans-plantation (day 0), recipients were sublethally irradiated at675 rad (total body irradiation) using an X-ray irradiator(RadSource 2000) at a rate of 106 rad/min (day 1). Intra-venous infusion of cells was performed in 0.2 mL PBS.

Cell isolation

Whole BMCs from wild-type or GFP-positive male donorswere harvested by flushing of medullar cavities of femur,tibia, and iliac bones. Single cell suspensions (5 · 108 cells)were loaded into the chamber of a counterflow centrifuge(Beckman Instruments) operating at a constant speed of3,000 rpm [40]. Fractions were collected in 200 mL at elutri-ation flow rate of 25 mL/min to isolate the smallest subset ofnucleated cells (Fr25), and the largest cells were collected inthe rotor off position. Fr25 cells were lineage-depleted byincubation at 4�C with rat-anti mouse monoclonal antibodies

(mAb) against CD5 (clone 53-7.3), GR-1 (clone RB6-8C5),Mac-1 (clone M1/70), B220 (clone RA3-3A1/6.1) extractedfrom hybridoma cell lines (ATCC), and purified TER119(eBioscience) [32]. Secondary goat-anti-rat antibodies conju-gated to magnetic beads (Dynal Biotech) were used to trapcells coated with primary antibodies in a magnetic field aspreviously described [40].

Flow cytometry

Measurements were performed with a Vantage SE flowcytometer (Becton Dickinson) on cells that underwent redcell lysis. Briefly, cells were suspended in lysis buffer (4.15 gNH4Cl, 0.5 g KHCO3, and 0.15 g disodium ethylenediami-netetraacetic acid) for 4 min at room temperature; lysis wasarrested with excess ice-cold medium followed by 2 washes.Hematopoietic chimerism was determined by GFP fluores-cence or mAb against minor antigens CD45.1 (clone A20;eBioscience) and CD45.2 (clone 104; eBioscience). In allmeasurements non-specific binding was prevented by addi-tion of 1mL mouse serum.

Tissue preparation

Mice were sacrificed by CO2 asphyxiation., and exsan-guination was performed by infusion of 30 mL of ice-coldPBS containing 1.5% fresh paraformaldehyde and 0.1% glu-taraldehyde through a catheter inserted in the left ventricle.Excised pancreata were placed in this medium for 2 h at 4�Cfor additional fixation, and immersed in 30% sucrose over-night [32]. Tissue embedded in OCT (Sakura Finetek) wasfrozen in isopentane suspended in liquid nitrogen and sec-tioned (3–6mm) with a Cryotome (Termo Shandon). Cryo-sections were stored at - 80�C before analysis.

Determination of blood insulin

Serum from NOD mice was collected by centrifugationand assessed in 96-well Microtiter Assay Plates (Millipore)using the Rat/Mouse Insulin ELISA Kit (R&D Systems).Absorbance at 450 and 590 nm was determined using anELISA PowerWave-10 in a plate reader (BioTeK). Insulinstandards were used to determine a calibration curve.

Immunofluorescence and fluorescencein situ hybridization

Immunofluorescence was designed according to severalconsiderations to allow detection of the donor cells withvariable GFP fluorescence intensities [40]. Donor cell analysisin host pancreata was performed in 2 steps to include im-munofluorescence in stained frozen sections and fluores-cence in situ hybridization (FISH) [32]. Sections were fixed inacetone and permeabilized by incubation with 0.2% Saponin,1% bovine serum albumin, and 0.1% Triton-100; werestained with primary antibodies for 1 h; washed; and coun-terstained with respective secondary antibodies for 30 min atroom temperature. Nuclei were labeled with Hoechst-33342(1:1,000; Molecular Probes), and sections were mounted inanti-fade medium (Dako) and air-dried. Serial cryosectionsfrom fixed pancreas were immunostained with primaryantibodies: polyclonal mouse-anti-proinsulin (1:20; R&DSystems), goat-anti-PDX-1 (1:5,000, kindly provided by

NEOGENESIS OF INSULIN PRODUCING CELLS FROM BONE MARROW 87

Shimon Efrat), rat anti-Pecam-1 (CD31, 1:100, CBL 1337,Chemicon-Millipore), biotinylated anti-mouse CD45 (1:100;Biolegend), biotinylated donkey-anti-mouse (1:1,000), andrabbit-anti-GFP (1:100; Santa Cruz Biotechnology). Theseprimary antibodies were counterstained with fluoresceinisothyocyanate-labeled donkey-anti-rabbit (1:200, JacksonImmunoresearch) and goat-anti-rat mAb (1:200; Santa Cruz),Alexa Flour 568-conjugated donkey-anti-goat (1:500), andCy3-conjugated rat-anti-goat (1:200; Molecular Probes).Biotinylated primary antibodies were counterstained withCy3-conjugated Streptavidin (1:400) and Cy5-conjugatedStreptavidin (1:500) both from Jackson Immunoresearch.

X and Y chromosomes were painted in fresh sections orimmunostained slides after treatment with 0.025% pepsin.Slides were rinsed with distilled water and 2 · saline sodiumcitrate (buffer) (SSC), dehydrated by sequential immersion in70%, 85%, and 100% ethanol at room temperature and air-dried. A second fixation in warm 70% formamide was fol-lowed by repeated dehydration in gradually increasing eth-anol concentrations. Nuclear probes (Applied SpectralImaging) denatured at 74�C (7 min) and 37�C (30 min) werehybridized at 37�C overnight, followed by sequential washingin 0.4 · SSC stringency solution at 74�C and 2 · SSC solutionsupplemented with 0.1% NP4O detergent at room tempera-ture. Washed slides were mounted with anti-fade containing4¢,6-diamidino-2-phenylindole. In our hands, Y chromosomewas detected using a painting probe in 77% – 14% of islet cellsin male controls (approximately 400 islet cells).

Images were acquired with an Axioplan 2 (C. Zeiss)fluorescence microscope equipped with an Apotome andwith a C. Zeiss confocal laser scanning microscope (LSM)using AxioVision 4.5 and LSM 510 software, respectively.Images were pseudocolored and RGB reconstructed usingAdobe Photoshop software.

Statistical analysis

Data are presented as means – standard deviations foreach experimental protocol. Results in each experimentalgroup were evaluated for reproducibility by linear regressionof duplicate measurements. Differences between the experi-mental protocols were estimated with a post hoc Scheffet-test and significance was considered at P < 0.05.

Results

The experimental model

A series of optimization experiments was performed todetermine the approach (Fig. 1A): (a) Five doses of 60mg/g

STZ administered on consecutive days results in sustainedhyperglycemia (400–500 mg/dL), and survival of C57BL/6mice without exogenous insulin supplementation. (b) Since itis unknown whether the grafted cells home first to the bonemarrow or the injured organ [41], the diabetic mice weresublethally irradiated at a dose of 675 rad. This combinedinjury causes severe damage to islets (Fig. 1B) and significant43% (16 of 37) mortality; however, the hyperglycemic micesurvived without exogenous insulin administration. (c)Analysis of the optimal temporal experimental sequenceshowed that infusion of the cells early after injury yields thebest glycemic control. (d) The smallest subset of BMCs wascollected by counterflow centrifugal elutriation (Fr25) anddepleted of cells expressing lineage markers (Fr25lin - ), re-sulting in a population largely positive for CD45 and nega-tive for GR-1 [40]. Mice with chemical diabetes infused withFr25lin - subset, which are not radioprotective [40], survivedprimarily through early recovery of endogenous hemato-poiesis after sublethal irradiation.

Fr25lin - cells home to the islets and amelioratethe course of chemical diabetes

Transplantation of 106 elutriated Fr25lin - cells into syn-geneic irradiated diabetic mice had a marked impact on thecourse of chemical diabetes. The cell transplant reduced peakblood glucose levels (P < 0.005) and caused significant controlof hyperglycemia after approximately 5 weeks (Fig. 1C). Themarkedly improved glycemic control over several months(P < 0.001 vs. STZ at 15 weeks), coinciding with the capacityto respond to glucose tolerance test (Fig. 1D) and elevatedinsulin levels (Fig. 1E) at the experimental end point. Thetime to partial reversal of hyperglycemia was longer in ourexperiments than reported for models that differed in theexperimental sequence, STZ doses, and types of grafted cells[10,11]; however, glycemic control was markedly improvedthan previously reported. At 7 months post-transplant sig-nificant numbers of donor cells were observed in the pan-creas; however, disruption of islet architecture and scatteredexpression of PDX-1 and proinsulin emphasize persistentinjury over extended periods (Fig. 1F).

To assess the patterns of cell migration to and incorpora-tion in the injured pancreas, male Fr25lin - CD45.2 + GFP +

donor cells were transplanted into female CD45.1 diabeticrecipients. The grafted Fr25lin - GFP + cells migrated effi-ciently to the injured islets, and GFPbright cells seeded pref-erentially in the vascular pedicle, ducts, and islet perimeter(Fig. 1G). Approximately 50% of the islet-homed cells ex-

FIG. 1. The experimental model and cell isolation. (A) Experimental design for induction of chemical diabetes with 5consecutive daily doses of 60 mg/g STZ, followed by sublethal TBI at 675 rad. Donor cells were identified by GFP expression,minor CD45 antigen disparity, and sex mismatch. Blood glucose was monitored and tissue was assessed by IHC and FISH atthe experimental end point. (B) Demonstrative immunohistochemical analysis of PDX-1 and proinsulin in islets at 1 weekafter combined STZ and radiation injury (scale bar 20mm). (C) Transplantation of Fr25lin - cells (n = 22) reduces significantlyblood glucose levels (P < 0.001) as compared to irradiated diabetic mice (STZ, n = 13). (D) Glucose tolerance test in naıve wild-type mice (n = 8), untreated mice (STZ), and recipients (n = 5) of Fr25lin - cells (n = 6) at 15 weeks after injury. (E) Serum insulinlevels at 15 weeks after chemical injury and transplantation (n = 3–4). (F) Representative analysis of islets at 7 months aftertransplantation of Fr25lin - GFP + cells demonstrates persistent injury and localization of donor cells in 4 islets (scale bar200 mm). (G) GFP + donor cells are found in the vascular pedicle, ducts, and surrounding the islets at 1 week post-transplant,and significant fraction of Fr25lin- cells are negative for the pan-hematopoietic marker CD45 (scale bar 40 mm). STZ, strep-tozotocin; TBI, total body irradiation; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; DAPI, 4¢,6-diamidino-2-phenylindole.

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88 ISKOVICH ET AL.

pressed CD45, possible mediators of immunomodulation[10,14,20,25], or other supportive mechanisms of islet re-covery from injury [11,13,17,24]. However, a significantfraction of Fr25lin - cells that migrated to the inner regions of

the islets were CD45-negative, suggesting participation innon-immunogenic mechanisms. One mechanism mediatedby CD45 - cells is the contribution of Fr25lin - GFP + cells toislet revascularization, affirmed by incorporation of GFP +

NEOGENESIS OF INSULIN PRODUCING CELLS FROM BONE MARROW 89

CD31 + cells in the vasculature [32] as reported for other cellsubsets [11,15,20]. Notably, endothelial differentiation oc-curred at 1–4 weeks post-transplantation, when Fr25lin - hada minor hematopoietic contribution, preceding in time thesubstantial improvement in glucose homeostasis.

Fr25lin - cells produce insulin

Delayed and gradual recovery of glucose homeostasisimplies that the partial resolution of hyperglycemia wascaused by differentiation of the grafted cells within theprocess of islet remodeling. To test this hypothesis we per-formed detailed analysis of phenotypic markers of b-cells:pancreatic and duodenal homeobox gene-1 (PDX-1) as anessential transcription factor involved in development of theendocrine pancreas [42], and proinsulin to prevent eventualuptake of insulin by the grafted cells from the environment[32]. This analysis revealed variable levels of GFP, PDX-1,and proinsulin in islet-incorporated Fr25lin - cells from GFPdonors (Fig. 2A), which might originate from inefficient de-tection of the reporter protein [32]. Juxtaposition of cytosolicGFPdim with nuclear PDX-1 in islet-resident cells was con-firmed by confocal microscopy (Fig. 2B), whereas the tran-scription factor was not detected in GFPbright cellssurrounding the islets. Abundant proinsulin expression inthese cells questions whether they are functionally deficientand unresponsive to systemic hyperglycemia, resulting inaccumulation of the hormone precursor. Intense staining forinsulin in donor cells (Fig. 2C) demonstrates their capacity toprocess the hormone precursor and make a true contributionto glycemic control.

Quantitative evaluation of donor cell incorporation

Since downregulation of GFP might be characteristic ofthe process of differentiation of the Fr25lin - cells towardadopting a phenotype compatible with insulin production[32], further identification of donor cells was performed us-ing a genomic marker in sex-mismatched transplants. Femalerecipients of male Fr25lin - cells displayed significant incor-poration of Y chromosome among islet cells staining positivefor PDX-1 (Fig. 3A). High-resolution analysis of 48 islets re-vealed the presence of Y chromosome and/or GFP donorcells in 38 islets, with significant variability in small islets ascompared with larger islets (Fig. 3B). An overall 209 donorcells positive for PDX-1 and/or proinsulin among 2,750evaluated b-cells indicate a 7.6% contribution to islet cellu-larity (Fig. 3C). In view of the challenges imposed by suchanalysis [32,43], this figure is a likely underestimate of dif-ferentiation of Fr25lin - cells.

Islet-resident Fr25lin - cells do not displayhematopoietic markers

Rigorous assessment of cell differentiation often imposesthe demonstration that stem cells or their myeloid progenyare not fusion partners of the injured b cells, causing lateraltransfer of specific markers rather than stem cells adoptingan endocrine pancreatic differentiation trait [44–47]. Themajority of grafted cells expressed CD45 (84% – 11%); how-ever, approximately 50% of islet-incorporated cells werenegative for this pan-hematopoietic marker (Fig. 1F). Ana-

lysis of sequential sections of the injured islets demonstratesCD45 and GR-1 expression in GFP + cells located at the isletperimeter, but not in cells located within the inner regions ofthe islets (Fig. 4A), consistent with non-hematopoietic phe-notypes of endothelial and insulin-producing cells. Fusion isexpected to result in a donor Y chromosome co-residing withseveral donor and host X chromosomes, whereas donor cellsdifferentiating to express PDX-1 in the inner islet regionsretained normal diploid XY genotype (Fig. 4B). A positivecontrol for these experiments is formation of syncitia com-posed of several GFP + CD45 + (and GR-1 + ) cells in proximityto the islets (Fig. 4C). The scattered appearance of proinsulinin these syncitial clusters most likely reflects clearance oftissue debris by immune cells at the islet perimeter. Althoughrare events of residual tissue fusion with donor stem cellsand their myeloid progeny may have occurred [48], we couldnot demonstrate such phenomena that might result in lateraltransfer of b-cell specific markers.

Discussion

This study presents evidence that the smallest nucleatedBMCs contribute to insulin production and ameliorateglycemic control over an extended period of time in amodel of murine chemical diabetes. Grafted cells identifiedby a genomic marker adopted phenotypic characteristic ofb-cells, produced insulin, downregulated GFP expressionand were found negative for determinants of hematopoi-etic lineages.

Previous reports have underlined participation of bonemarrow-derived cells in remodulation of the islets after in-jury. As descendents of a common progenitor, hematopoieticand endothelial precursors residing in the bone marrowsupport tissue revascularization and contribute to neo-vascularization [10,11,14,15,20]. In addition, local im-munomodulation achieved by mesenchymal stromal cells[19,25], one [14] or multiple BMC infusions [17], or concur-rent bone marrow mobilization and induction of hemato-poietic differentiation [10,20], support tissue remodelingafter injury through b-cell expansion [11,49] and/or activa-tion of tissue progenitors [2–8]. Mesenchymal stromal andimmuno-hematopoietic cells support islet recovery also byremodulation of the stroma and provision of growth factors[18,1,22]. Direct contribution to neovascularization by in-corporation of GFP + CD31 + cells in islet vasculature is evi-dence of developmental versatility of the Fr25lin - cells usedin this study. However, the various functions attributed todifferent cell types derived from hematopoietic compartmentswere largely inefficient in reinstitution of glucose homeo-stasis over extended periods [9–25].

Modest contributions of various hematopoietic cells toislet cellularity have been reported, ranging from incidentalincorporation [10,19,22,23,26,50,51] to 3% after myeloablativebone marrow transplantation [12] and 2.5% GFP + insulin +

cells negative for PDX-1 after chemical injury [11]. In con-trast, Fr25lin - cells migrated efficiently to the injuredpancreas, with particular affinity to the islets, ducts, andvascular walls, and improved significantly glycemic control:CD45-negative cells that infiltrated the islets upregulatedPDX-1 and produced proinsulin. Detection of proinsulin wasselected to prevent an experimental bias caused by uptake ofinsulin from the injured b-cells [32]. Accumulation of the

90 ISKOVICH ET AL.

hormone precursor in the differentiated donor cells mightindicate inefficient conversion to insulin, or underdevelopedglucose sensing machinery [52,53]. The proven capacity ofdonor cells to convert proinsulin into insulin, and elevatedserum insulin levels in mice that recovered normoglycemia

demonstrate neogenesis of competent cells that produce andsecrete the hormone. Reconstitution of 7.6% functionalinsulin-producing cells originating from the male donorFr25lin - stem cell-enriched fraction is a substantial mecha-nism of stabilization of glucose homeostasis. Participation of

FIG. 2. Islet-resident GFPdim donor cells produce insulin. (A) Exclusion of the GFPbright cells from the analyzed fieldfacilitates analysis and allows identification of GFPdim cells expressing proinsulin and PDX-1 (yellow arrowheads). White arrowpoints to sequestration of GFP and proinsulin in different intracellular compartments (scale bar 10mm). (B) Z-stack imagesacquired at 0.5 mm intervals with a confocal microscope demonstrate juxtaposition of nuclear PDX-1 and cytosolic GFPdim, tosubstantiate their presence in the same cell. A GFPbright cell is negative for PDX-1 (scale bar 10 mm). (C) GFPdimPDX-1 + donorcell displays abundant cytosolic insulin, emphasizing the capacity of these cells to process proinsulin. Intense staining forinsulin overrides the faint GFP expression (scale bar 5mm).

NEOGENESIS OF INSULIN PRODUCING CELLS FROM BONE MARROW 91

donor cells in islet regeneration is most likely under-estimated, considering the technical difficulties in combinedIHC and FISH analysis, the physiological downregulationof GFP expression in differentiating progenitors, and theanticipated silencing in endocrine cells within the islets[32,54]. Nevertheless, neogenesis of endothelium andinsulin-producing cells affirms the developmental plasticity

of this subset of adult bone marrow-derived stem cells inaddition to conversion into hepatocytes and various epi-thelia [37–39].

The time frame for differentiation into insulin-producingcells reported here is longer than previously observed in theliver [38] and other epithelial tissues [39], and is shorter thanthe contribution of these cells to long-term hematopoietic

FIG. 3. Identification of donor cells by Y chromosome and quantitative analysis. (A) Representative image of islet-infil-trating cells at 16 weeks after sex-mismatched transplantation of Fr25lin - cells (male/female). The image was reconstructedwith IHC layers for PDX-1 and DAPI (scale bar 10 mm). Superposition of FISH analysis for donor Y chromosome in 7 islet-infiltrating cells at high magnification (insets). (B) Analysis of donor cell distribution as a function of islet size showed quitestable levels of incorporation. (C) Estimation of incorporation of donor cells that expressed PDX-1 and proinsulin wasevaluated at 10–16 weeks post-transplantation in 48 islets from 7 recipients.

92 ISKOVICH ET AL.

FIG. 4. Insulin-producing donor cells lack hematopoietic markers and are not products of fusion. (A) Two consecutive sectionswere stained for CD45 (upper panels) showing extra-islet location of immuno-hematopoietic GFPbrightCD45+ donor cells (yellowcircles), which are negative for PDX-1 and proinsulin (lower panels). Donor cells within the islet are GFP+ CD45- (white circles, scalebar 20mm). (B) Consecutive sections were evaluated for endocrine markers and genomic markers. Upper panels present FISHanalysis of male Fr25lin - donor cells (Y chromosome) and IHC for GFP and PDX-1 (scale bar 10mm). Lower panels present high-resolution FISH analysis of paired X and Y chromosomes in PDX-1+ cells (inset). (C) Morphology of debris clearance isdemonstrated by a rare observation of a number of CD45-positive cells engulfing particles staining positive for proinsulin. Scalebar 20mm. Enlarged inset: the scattered appearance of proinsulin at an extra-islet location is characteristic of cell degradation.

NEOGENESIS OF INSULIN PRODUCING CELLS FROM BONE MARROW 93

reconstitution [34–36,40]. It is conceivable that restriction ofimmuno-hematopoietic differentiation is strongest in multi-potent stem cells residing in the marrow space. Accordingly,effective hematopoiesis from Fr25lin - cells occurs lastamong the various non-hematopoietic differentiation traitsadopted by these adult bone marrow-derived stem cells[38,39]. The bone marrow is a likely reservoir of repair unitsbecause they can be readily distributed to sites of injury viathe systemic circulation. Fr25lin - cells are not only physio-logical residents of the bone marrow, but are endowed withparticular affinity to this compartment, and can be enrichedusing an in vivo homing assay [41]. Further, these cells dis-play several significant characteristics of primitive (stem)cells: quiescent mitotic state early after transplantation[36,40], self-renewal capacity emphasized by indefinitemultilineage hematopoietic reconstitution [34–36,40], andfunctional contribution to multiple tissue, including endo-thelium, endocrine pancreas, liver [37], and epithelia [39]. Inaddition, these cells display glial and neuronal markerswhen implanted in models of retinal injury in the absence ofradiation, questioning whether these cells require initialhoming to and priming in the bone marrow (N. Goldenberg-Cohen, unpublished data). We do not exclude the possibilitythat the Fr25lin - and Fr25lin - PKHbright subsets includethe CD45 - SCA-1 + CXCR4 + lin - very small embryonic-like(VSEL) cells [51], which is consistent with detection ofCD45 - donor cells within the injured islets in this and priorstudies [19,26,50]. However, unlike VSEL, most of the graf-ted cells are CD45-positive, and depletion of SCA-1 + subsetsis unrelated to functional islet reconstitution by Fr25lin - cells(S. Iskovich, unpublished data). Furthermore, molecularanalysis of the elutriated Fr25lin - subset shows low levelexpression of mRNA and protein of determinants of theendocrine pancreatic differentiation trait (N. Goldenberg-Cohen, unpublished data), negating the possibility of thepresence of committed tissue progenitors within the fractionof BMCs used in this study [55,56].

Despite the rightful criticism raised by negative results[9,32,57,58], we report failure to detect fusion events. Bothstem cells and their myeloid progeny have been shown toadopt parenchymal phenotypes through lateral transfer ofmarkers caused by cell fusion [44–47]. We could demonstrateneither pluriploid X chromosomes in the PDX-1 + proinsulin +

donor cells consistent with stem-parenchyma chimerism, norexpression of CD45 or GR-1 consistent with uptake of b-cellmarkers through fusion or phagocytosis. The possibility thatfusion can reconstitute tissue function remains to be deter-mined in view of the very low frequency of these events [48].According to the incidence reported in a fusogenic organsuch as the liver [44–47], it is estimated that fusion wouldaccount for 10 - 2–10 - 3 of the 7.6% donor cells producinginsulin, and therefore is a quite insignificant mechanism.

The possibility to intervene in the process of islet re-modeling and design regenerative approaches is a tangibleperspective. Somatic pancreatic cells have been reverted toand endocrine phenotype by expression of few transcriptionfactors [59] and interconversion of cell types within the isletshas been achieved by regression to a more primitive stateand redirection [60]. Our current data demonstrate robustneogenesis of insulin-producing cells from adult bonemarrow-derived primitive cells in vivo, which can stabilizeglycemic control. In addition to the capacity of non-

myeloablative bone marrow transplantation to arrest auto-immune insulitis [61], enriched autologous populations ofprimitive cells have the potential to regenerate the tissue.

Acknowledgments

We thank Dr. Saul Sharkis and Dr. Michael Collector forthe outstanding support, discussion, and conceptual contri-bution to this study. Funding was provided by a generousgrant from the Leah and Edward M. Frankel Trust for bonemarrow transplantation.

Author Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Prof. Nadir Askenasy

Frankel LaboratoryCenter for Stem Cell Research

Schneider Children’s Medical Center of Israel14 Kaplan Street

Petach Tikva 49202Israel

E-mail: anadir@012.net.il

Received for publication February 02, 2011Accepted after revision April 01, 2011

Prepublished on Liebert Instant Online April 4, 2011

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