Stem Cell Research 15 (2015) 299–304

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Short report

Novel source of human hematopoietic stem cells from peritonealdialysis effluents

Jinhui Shen a, Junke Zheng b, Ramesh Saxena c, ChengCheng Zhang b,⁎⁎, Liping Tang a,d,⁎a Department of Bioengineering, University of Texas at Arlington, PO Box 19138, Arlington, TX 76019, United Statesb Departments of Physiology and Developmental Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, United Statesc Division of Nephrology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX 75390, United Statesd Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan

⁎ Correspondence to: L. Tang, Department of BioengiArlington, PO Box 19138, Arlington, TX 76019, United Sta⁎⁎ Correspondence to: C.C. Zhang, Departments of PBiology, University of Texas Southwestern Medical CeDallas, TX 75390, United States.

E-mail addresses: Alec.Zhang@UTSouthwestern.edu (C(L. Tang).

http://dx.doi.org/10.1016/j.scr.2015.07.0031873-5061/© 2015 The Authors. Published by Elsevier B.V

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 March 2014Received in revised form 9 July 2015Accepted 14 July 2015Available online 16 July 2015

Keywords:Peritoneal dialysisHematopoietic stem cells

Hematopoietic stem cells (HSCs) hold great promise for the treatment of various diseases and blood disorders.However, limited availability of these cells has hampered their applications in clinical and biological research.Here we have identified a new source of autologous human HSCs in peritoneal dialysis (PD) effluents from pa-tients with end stage renal diseases (ESRDs). Cells isolated from PD effluents contain a Lin−/CD34+/CD38−/CD90+ sub-population and can repopulate NOD/SCID/gamma−/− mice in serial transplantation. Differingfrom cord blood HSCs, PD-derived HSCs have high tendencies to repopulate peritoneal cavity and spleen withmyeloid cells and B lymphocytes. Repopulating HSCs also reside in peritoneal cavities in mice. The isolation ofHSCs from peritoneal cavities provides a novel and promising source of autologous and functional HSCs forstem cell research and possible clinical use.

© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).

1. Introduction

Hematopoietic stem cells (HSCs), defined by their ability to self-renew and differentiate into all blood cell types (Till and Mc, 1961;Seita and Weissman, 2010), have been isolated from bone marrow(Mathe et al., 1963), mobilized peripheral blood (Gianni et al., 1989),and umbilical cord blood (Gluckmanet al., 1989). They hold great prom-ise for the treatment of many diseases, including selected hematologicmalignancies, immunodeficiencies, hemoglobinopathies, bone marrowfailure syndromes, and congenital metabolic disorders (Bryder et al.,2006). Previous studies indicate that, during development, hematopoi-esis takes place in a variety of sites including yolk sac, aorta–gonad–mesonephros, liver, spleen, and bone marrow. In adulthood, HSCsmainly reside in the bone marrow, and also exist in extramedullaryorgans including spleen and liver, especially during immune responsesfollowing infection or during failed marrow hematopoiesis (Kim,2010). Identification of additional sources of HSCs will further ourunderstanding of the biology of HSCs and the interaction between

neering, University of Texas attes.hysiology and Developmentalnter, 5323 Harry Hines Blvd,

. Zhang), ltang@uta.edu

. This is an open access article under

stem cells and their microenvironment, and lead to the developmentof new strategies of HSC-based therapies.

Our recent studies have revealed that subcutaneous implantation ofbiomaterials promotes the recruitment of HSC to the implantation sites(Nair et al., 2011). Although themechanism(s) of biomaterial-mediatedstem cell recruitment is unclear, it is likely that inflammatory productsreleased during foreign body reactions prompt the recruitment ofHSCs. To test whether such observations also hold true in humans,we examined patients with end stage renal disease (ESRD) whowould undergo peritoneal dialysis (PD). Since PD patients have a cath-eter implanted in the peritoneum prior to the treatment, we testedwhether dialysate effluents from incident PD patients contained HSCs.By analyzing cells isolated from PD effluents following catheter implan-tation, we identified a Lin−/CD34+/CD38−/CD90+ sub-population ofcells in PD effluents. The hematopoietic multipotency of PD effluent-isolated HSCs was assessed using a transplantation assay. Concordantwith this result, we also identified functional HSCs from mouse perito-neal cavity.

2. Materials and methods

2.1. Study population

Peritoneal dialysis (PD) fluid samples were collected from 49 ERSDpatients visiting the University of Texas Southwestern Medical Center/DaVita PD Clinic (Irving, TX) within a week after catheter implantation

the CC BY license (http://creativecommons.org/licenses/by/4.0/).

300 J. Shen et al. / Stem Cell Research 15 (2015) 299–304

for post-operative catheter care and PD training. For PD procedure thecatheter was flushed with 1–2 l of dialysate solution (2.5% Dianeal PDsolution, Baxter, Deerfield, IL), and the effluent was collected for exper-iment at the PD clinic after each cycle. The protocol of PD effluent collec-tionwas approved by the Institutional Review Board of theUniversity ofTexas Southwestern Medical Center at Dallas. Patients with peritonitiswere excluded from this study. Of all 49 patients, 12 had been on hemo-dialysis (HD) for different periods of time prior to commencement ofPD, while 37 patients had no prior HD treatment. The demographicinformation of the patients is listed in Table 1.

2.2. Flow cytometry analyses of human PD effluent samples

For flow cytometry analysis, some of the recovered cells werefollowed by lysis of RBC using RBC lysing buffer (Sigma Chemical Co.,St Louis, MO) according to the manufacturer's instructions. For RBClysis, 5 ml of RBC Lysing Buffer was gently mixed and incubatedwith the cell pellet collected from a patient effluent at room tempera-ture for 10 min. The mixture was diluted with 20 ml of mediumsupplemented with FBS, and the cells were collected after centrifuga-tion at 250–500g for 7 min. Cells were then washed in PBS, countedand adjusted to 5 × 106/ml in ice cold PBS with 2% FBS, and stainedwith monoclonal antibodies anti-human APC-CD90, FITC-CD34, PE-CD38 (BD Bioscience, San Jose, CA), Biotin-Lineage cocktail (MiltenyiBiotec Inc, Auburn, CA), and PE-Cy5.5-Strepavidin secondary antibody(BD Bioscience, San Jose, CA) for HSCs staining. PE-CD3, PE-CD19, PE-CD20, FITC-CD15, FITC-CD66b, PE-CD45, and PE-CD71 (BD Bioscience,San Jose, CA) were also used for hematopoietic lineage staining.FITC-CD34, PE-CD38 (BD Bioscience, San Jose, CA), Biotin-Lineage kit(Miltenyi Biotec Inc, Auburn, CA), PE-Cy5.5-Strepavidin secondary anti-body (BD Bioscience, San Jose, CA), APC-CD10, eFluo 450-CD45RA, andAPC-CD123 were used for progenitor cell staining. Stained cells wereanalyzed on BD FACSCalibur or FACSAria (BD Bioscience, San Jose,USA) for the presence of various hematopoietic populations.

Table 1Demographic characteristics of peritoneal dialysis patients.

Patient characteristics Number

Male 28Females 21Total 49Median age (years) 52Dialysis historyPrevalent patients 0

RaceAfrican American 24Caucasian 10Hispanics 12Asian/Pacific Islander 3

Prior hemodialysis among incident patientsPrior hemodialysis 12No prior hemodialysis 37

Causes of end stage kidney diseaseDiabetes mellitus 22Hypertension 12

Primary glomerulonephritisIgA nephropathy (1)Chronic glomerulonephritis (1)

2

Lupus nephritis 2Autosomal dominant PKD 4HIV nephropathy 2Failed Kidney transplant 2Chronic interstitial nephritis 0Hepatitis C associated nephropathy 2Unknown cause 1

2.3. In vivo repopulation in NSG mice

For the repopulation study, non-obese diabetic (NOD)-scid IL2rγnull

(NSG) mice were purchased and maintained at the University of TexasSouthwestern Medical Center animal facility. All animal experimentswere performedwith the approval of University of Texas SouthwesternCommittee on Animal Care.

To study thehematopoieticmultipotencyof the cells derived fromPDeffluents, freshly recovered peritoneal cells (1–10×107 cells per animal)were injected intraperitoneally or intravenously via the retro-orbitalroute into sub-lethally irradiated (2.5 Gy) 8- to 10-week-old NSG mice.Eight weeks after transplantation, bone marrow cells from the recipientmice were analyzed by flow cytometry for the presence of human cells.Monoclonal anti-human PE-CD45, PE-CD71, FITC-CD15, FITC-CD66b, PE-CD19, PE-CD20, Biotin-CD3, APC-streptavidin secondary antibody, andFITC-CD34 antibodies (BD Bioscience, San Jose, CA) were used for thestaining of human myeloid, lymphoid, and primitive cells (Zhang et al.,2008). To compare different repopulation rates in organs, peritoneal la-vage cells and spleen cells were also isolated from a few NSG recipientmice for lineage staining.

To evaluate long term reconstituting potential of PD derived HSCs,bone marrow aspirates from one hind leg of a primary recipient, orperitoneal lavage cells, or spleen cells from a primary recipient weretransplanted into a secondary recipient (Zhang et al., 2008; Hoganet al., 2002). For limiting-dilution analysis, mice were consideredpositive for human HSC engraftment when at least 1% (for primarytransplantation) or 0.1% (for secondary transplantation) CD45/71human cells were detected among the mouse bone marrow cells(Zhang et al., 2008).

2.4. Hematopoietic colony assays

Freshly isolated peritoneal cells followed by RBC lysis were washedin PBS and diluted to 1 × 106/ml in Iscove's modified Dulbecco'smedium (IMDM) with 2% FBS, and then seeded into methylcellulosemedium H4436 (StemCell Technologies) for CFU-GEMM, CFU-GM, andBFU-E colony formation, according to the manufacturer's protocols(Zheng et al., 2011a; Zhang et al., 2006).

2.5. EGFP transgenic mice peritoneal cell transplantation and repopulationstudy

For the mice peritoneal cell transplantation study, EGFP negativeC57BL/6 mice (6–8 weeks old) were used and maintained at theUniversity of at Arlington animal facility. The animal use protocol wasapproved by the Institutional Animal Care and Use Committee ofthe University of Texas at Arlington. EGFP negative C57BL/6 mice(6–8 weeks old) were irradiated (whole body X-ray irradiation) at1000 cGy and then transplanted with sex matched peritoneal cells iso-lated from EGFP transgenic mice through retro-orbital injection. Eightweeks after transplantation, peripheral blood and peritoneal cells ofrecipient mice were isolated for flow cytometry analysis for GFP+

hematopoietic lineage markers Thy1.2, B220, Mac-1, and Ter119 essen-tially as we described (Kang et al., 2015). Eighteen weeks after trans-plantation, peripheral blood, peritoneal cells, bone marrow cells, andspleen cells of recipient mice were collected for GFP+ lineage markeranalysis again to check long-term peritoneal HSCs.

2.6. Statistical analysis

Data are expressed as Mean ± SEM. IBM SPSS Statistics 19 softwarewas used for analysis. Significant levels were calculated using student'st-test. One-way ANOVA and post-hoc Scheffe's test were used forcomparisons between multiple groups. Differences were consideredsignificant when p b 0.05.

Fig. 1. Characterization of hematopoietic cells isolated from PD effluents. Representative flow cytometry plots reveal the presence of (A) Lin−/CD34+/CD38−/CD90+cells, N = 140,(B) CMP (Lin−/CD34+/CD38+/IL3Ralow/CD45RA−), MEP (Lin−/CD34+/CD38+/IL3Ra−/CD45RA−), GMP (Lin−/CD34+/CD38+/IL3Ralow/CD45RA+), and CLP (Lin−/CD34+/CD38+/CD10+) cells in a PD sample, N = 5. (C) The frequencies of various hematopoietic populations and CD3+T lymphocytes, CD19+/CD20+ B lymphocytes, CD15+/CD66b+ myeloid cells,and CD71+ erythroid cells in PD effluent cells were also determined. N = 5. (D–F) The numbers and frequencies of Lin−/CD34+/CD38−/CD90+ cells in PD effluents recovered from 49patients at different times (0–100 days, N = 140 for D/E and N = 120 for F) following peritoneal catheter implantation were quantified using flow cytometry. Vertical lines denote±1 SEM.

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3. Results and discussion

By analyzing surface markers of cells isolated from PD effluents, weidentified a Lin−/CD34+/CD38−/CD90+ (~0.14±0.03%) subpopulation(Fig. 1A), which are known to enrich for human HSCs. PD cells alsocontain phenotypic hematopoietic progenitors including commonmye-loid progenitors (CMP), megakaryocyte–erythroid progenitors (MEP),granulocyte/monocyte progenitors (GMP), and common lymphoid pro-genitors (CLP) (Fig. 1B). The overall peritoneal cells further includeCD3+T-lymphocytes, CD19+/CD20+B-lymphocytes, CD15+/CD66b+

myeloid cells, and CD71+ erythroid cells (Fig. 1C). After analyzing allsamples, we found that the phenotypic HSCs were consistently presentin the PD effluents isolated from different patients at different times(Fig. 1D–F). Statistically, analyses revealed that a patient's gender, age,

prior hemodialysis (HD) treatment and duration following cathetertransplantation had no influence on the numbers of Lin−/CD34+/CD38−/CD90+ cells found in the PD effluents. The existence of hemato-poietic progenitors was also confirmed by colony formation assays(Supplemental Fig. 1) (Zheng et al., 2011b, 2012). Additional analysesrevealed that, on average, about 5.6 × 104 Lin−/CD34+/CD38−/CD90+

cells can be recovered from one PD effluent sample. There were onaverage 2.8 × 105 phenotypic HSCs acquired from5 effluents of each pa-tient during the PD training period. The frequency and number of HSCsacquired from PD effluents and patients varies greatly (SupplementalFig. 2). The reason(s) for such variations remain undetermined.However, statistical analyses have excluded the influence of prior he-modialysis, gender, and ages on PD-derived HSC numbers. In addition,the numbers of phenotypic HSCs recovered from PD effluents did not

Fig. 2. Human PD-derived HSCs are capable of repopulating myeloid and lymphoid lineages in NSG mice. (A) Representative flow cytometry plots show multi-lineage repopulation ofdonor human cells including monoclonal anti-human CD45+/CD71+ human hematopoietic cells (abbreviated as Total), CD15+/CD66b+ myeloid cells (Mye), CD19+/CD20+/CD34− Blymphocytes (B-Lym), and CD34+ primitive cells (Pri). (B) The percentage of different types of human hematopoietic cells in the recipient mice bone marrow were quantified 8 weeksafter human peritoneal cell implantation. N = 9. (C) Bone marrow cells isolated from bone marrow of primary transplanted mice were used for secondary transplantation. Differenttypes of human hematopoietic cells in mouse bone marrow following secondary transplantation were quantified. N = 5. Vertical lines denote ±1 SEM.

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significantly reduce for up to 100 days after surgery. These resultssuggest that HSCs are either resident cells or continuously replenishedin the peritoneal cavity.

The functionality of these phenotypic HSCs isolated from PDeffluents was evaluated by transplantation into sub-lethally irradiatedNOD/SCID/gamma−/− (NSG) mice (Zheng et al., 2012). Fig. 2Ashows multi-lineage engraftment of total hematopoietic (CD45/71+),myeloid (CD15/66b+), B-lymphoid (CD34−CD19/20+), and primitive(CD34+) human cells. Collectively, for 107 donor cells, there were2.87 ± 1.66% total human hematopoietic cells, 0.80 ± 0.70% myeloidcells, 0.19 ± 0.09% lymphoid cells, and 0.92 ± 0.53% primitive cells inrecipient bone marrow (Fig. 2B), attesting the existence of functionalHSCs in the donor population. To assess self-renewal and long-termfunctionality of PD-derived HSCs, we collected bone marrow cellsfrom the primary mice and then transplanted them into sub-lethallyirradiated secondary recipients. The donor-derived cells showedcomparable positive engraftment of B-lymphoid cells (0.97 ± 0.54%) inbone marrow as in the primary recipients, and lower engraftment ofmyeloid cells (0.070 ± 0.031%) and primitive cells (0.019 ± 0.007%)(Fig. 2C). Together, our results imply that PD-derived HSCs containrepopulating HSCs. Finally, the transplantation routes (intraperitonealand intravenous injection) have no effect on the final outcome ofchimerisms.

To investigate whether PD-derived HSCs behave differently fromHSCs from other sources, we compared the engraftment of 107 humanPD cells and 105 human cord blood (CB) CD34+ cells in differenttissues/organs of the recipient mice via intravenous injection. At 2months post-transplantation, the indicated number of CB mononuclearcells had a greater engraftment in the recipient bone marrow, whereas

Fig. 3. Comparative study of the repopulation and differentiation capability of PD-derived HSCisolated from different sources, PD-derived HSCs (labeled as “Human PD donor”) and cord blomice. At 2months post-transplantation, bonemarrow cells from the transplanted animals werecentages of different hematopoietic lineage cells in different organswere compared between difare found in higher percentages than those in bone marrow. N = 8. (C–D) Functional charactperitoneal cells were isolated from EGFP transgenic C57BL/6 mice and mixed with 105 GFP-Cthrough retro-orbital injection. Cells isolated from themice peripheral blood (labeled as “PB”) aof hematopoietic cells, including B220+ B lymphocytes, Mac-1+myeloid cells, Thy1.2+ T lymphN = 6. (E) Representative flow cytometry plots showing the GFP+ compartment contained m

the PD cells repopulated better in the peritoneal cavity and spleen, andproduced higher percentages of myeloid cells and B-lymphocytes thanCB counterparts (Fig. 3A). We suspect that, after residing in the perito-neal cavities, PD-derived HSCs are “primed” to initiate adaptive andinnate immune responses against potential invasion of pathogensthrough intestinalmucosa. It is also possible that PDHSCs have a certaindisadvantage while homing to recipient bone marrow. While we didcarry out intrafemoral transplantation, this type of injection can onlyadminister a volume less than 20 μl. This small injection volume is insuf-ficient for PD cells to produce detectable reconstitution. To explain the“primed”properties,we believe that donor PDHSCs have distinct intrin-sic potential asc compared to bonemarrow or cord blood HSCs.We alsosuspect that the recipient peritoneal and extramedullary sites provideunique microenvironment for PD HSCs to home and differentiate.

To investigate whether PD-derived HSCs exist in other mammals,we analyzed the resident cells in the peritoneal cavity of C57BL/6mice without any prior treatment. We found that there was a higherratio (0.51 ± 0.04%) of Lin−/Sca1+/c-kit+ population in the mouseperitoneal cavity than in bonemarrow (0.05 ± 0.01%) (Fig. 3B). Similarobservations were also noted in Balb/C mice. These results support thatPD derived HSCs may reside in the rodent peritoneal cavity. We believethat amuch higher frequency of peritoneal cavity HSCs than BMHSCs iscontributed by the fact that the total cell number in peritoneal cavity islow (~106 cells per mouse). Because infection may frequently occur inthe peritoneal cavity, a high frequency of HSCs in the peritoneal cavitymay help to fight infection by timely differentiation into differenttypes of hematopoietic and immune cells.

To characterize their function and repopulation capability, HSCs iso-lated from EGFP+ C57BL/6 mice' peritoneal cavities were transplanted

s and cord blood HSCs, and mouse peritonea HSCs. To investigate the responses of HSCsod HSCs (labeled as “Human CB donor”) were transplanted in sub-lethally irradiated NSGrecovered and then analyzed for the presence of human hematopoietic cells. (A) The per-

ferent sources of cells. N=4. (B) Resident Lin−/Sca-1+/c-kit+HSCs in themouse peritoneaerization of mouse peritoneal HSCs was carried out using reconstitution analysis. 5 × 106

57BL/6 bone marrow cells, and was then transplanted into each irradiated C57BL/6 micend peritoneal dialysis (labeled as “PD”) were analyzed for the presence of different lineagesocytes, and Ter119+ erythroid cells, at 8weeks (C) and (D) 18weeks post-transplantation.ulti-lineage repopulating cells. Vertical lines denote ±1 SEM.

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into lethally irradiated wild type C57BL/6 mice through retro-orbitalinjection. Eight weeks later, in GFP+ repopulated cells, we found signif-icantly higher percentages of B-lymphocytes (55.73±1.97% vs. 31.82±7.54%) and macrophages (61.23 ± 4.30% vs. 18.44 ± 12.11%) in theperitonea than in the peripheral blood (Fig. 3C). There were higher per-centages (43.54 ± 6.04% vs. 9.09 ± 1.20%) of T-lymphocytes in the pe-ripheral blood than in the peritonea. Further analyses were carried outon the same animals after implantation for 18 weeks. The results re-vealed similar trends of cell distribution in which substantially higherpercentages of B-lymphocytes (43.06 ± 3.47% vs. 29.33 ± 0.71%) andMac-1+ myeloid cells (42.43 ± 3.83% vs. 14.25 ± 3.46%) were foundin the peritonea than in the peripheral blood (Fig. 3D). This bias ofperitoneal cavity HSCs to differentiate into B cells or macrophagesmay be beneficial to immune actions in response to infections. Thepercentages of erythroid cells were slightly higher in peripheral bloodthan in the peritonea. The mouse peritoneal HSCs possess propertiesdifferent from peripheral HSCs or inflammatory cells. We used 105 ofdonor peripheral blood mononuclear cells from CD45.1 C57BL/6 miceand performed a similar competitive reconstitution analysis, and didnot observe donor engraftment in CD45.2 C57BL/6 recipient mice.HSCs from other periphery inflammatory sites such as tumor stromaalso have much lower repopulation activity (Huynh et al., 2011), com-pared to peritoneal HSCs. These results support that mouse peritoneacontain repopulating HSCs, and peritoneal HSCs have great potential todifferentiate into B-lymphocytes and macrophages in the peritonea. It isplausible that due to frequent infections in the peritoneal cavity, a highfrequency of HSCs on site will help timely differentiation into differenttypes of hematopoietic and immune cells to combat infections. In accor-dance with the fact that there exist myeloid- and lymphoid-biased sub-populations of HSCs (Muller-Sieburg et al., 2002; Oguro et al., 2013;Beerman et al., 2010; Dykstra et al., 2007; Kent et al., 2009; Challenet al., 2010; Morita et al., 2010; Ema et al., 2005; Benveniste et al.,2010), we suspect that the donor peritoneal HSCs have distinct intrinsicpotentials than bone marrow or cord blood HSCs. We also suspect thatthe recipient peritoneal and extramedullary sites provide a uniquemicro-environment for peritoneal HSCs to home and differentiate. Future com-parison of properties of peritoneal and bone marrow HSCs in geneexpression, cell fates, immune privilege, and metabolic feature (Zhenget al., 2011b, 2011c; Zhang, 2012; Zhang and Sadek, 2014; Simsek et al.,2010) are warranted. We believe that peritoneal HSCs may serve as apromising source of autologous HSCs, and efforts to utilize these cells,as in stem cell banking, may prove beneficial to stem cell research andclinical treatment.

Acknowledgments

This work was supported by the NIH RO1 GM074021,EB007271, CA172268, P30DK079328, CPRIT 140402, Leukemia &Lymphoma Society Awards 260071 and 6024–14, March of DimesFoundation Award #1-FY14-201, and Robert A. Welch Foundationgrant I-1834.

AuthorshipContribution: J.S. and J.Z. performed the experiments; R.S.

coordinated the collection of human samples; all authors designed thestudy and wrote the paper.

Conflict of interest disclosureThe authors declare no competing financial interests.

Appendix A. Supplementary material

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.scr.2015.07.003.

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