Human Umbilical Cord Blood–Derived Stromal Cells: A New Resource in Hematopoietic Reconstitution in Mouse Haploidentical Transplantation

Human Umbilical Cord Blood–Derived Stromal Cells: A New Resource in Hematopoietic Reconstitution in Mouse Haploidentical Transplantation C. Zhang, X.-H. Chen, X. Zhang, L. Gao, P.Y. Kong, X.G. Peng, X. Liang, L. Gao, and Q.Y. Wang ABSTRACT Objective. Our previous study showed that human umbilical cord blood– derived stromal cells (hUcBdSCs) expanded CD34⫹ cells in vitro. This study further explored the role of hUcBdSCs in vivo. Methods. The cultured hUcBdSCs were infused into transplanted haploidentical mice to observe hematopoietic recovery and complications. Results. The engraftment was faster in transplantation with hUcBdSCs than without hUcBdSCs. The numbers of fibroblast (CFU-F), granulocyte/monocyte (CFU-GM), erythrocytic (CFU-E), and megakaryocyte (CFU-Mg) colony-forming units were greater among mice transplanted with hUcBdSCs than without hUcBdSCs. The scoring of graft-versus-host disease was significantly lower in mice that had been subjected to transplantation with hUcBdSCs than without hUcBdSCs. The infused hUcBdSCs migrated to the bone marrow of the recipients. Conclusions. These data indicated that hUcBdSCs improved hematopoietic reconstitution in haploidentical transplantation in mice. llogeneic hematopoietic stem cell transplantation (alloHSCT) is an effective curative therapy for a variety of hematologic malignancies.1,2 Genotypically HLA-identical sibling donors, who are available for about 30% of caucasian patients,3 are still regarded to be the best donors for allo-HSCT. However, most patients (⬃70%) do not have an HLA-matched donor. The use of hematopoietic stem cells (HSCs) from relatives, who are partially matched for HLA, has advantages for patients lacking HLA-matched sibling donors or fully matched unrelated donors. All patients have at least one HLA-partially matched family member, parent, sibling, or child, who is immediately available to serve as a donor. However, the use of mismatched allografts has been disappointing due to the high incidence of graft-versus-host disease (GVHD) and infectious complications resulting in a high transplant-related mortality (TRM) and overall mortality.2 The data from HLA-haploidentical transplantation indicate that both graft failure and GVHD remain as problems. The fact that there were only a few survivors as a result of this therapy led to the closure of many trials.4 The development of new strategies or graft manipulations aimed at improving engraftment and of a better tolerated, less toxic, lower dose-conditioning regimen have become

A

critical research areas in HLA-haploidentical transplantation. The hematopoietic inductive microenvironment (HIM) is the site of origin, proliferation, differentiation, and development of HSCs. As an important component of HIM, stromal cells are related to the self-renewal, proliferation, differentiation, and homing of HSCs, as well as the occurrence, progression, and prognosis of hematologic diseases.5–11 Earlier research into stromal cells and their applications has focused on bone marrow stromal cells.12–17 However, it is also important to study human umbilical cord blood– derived stromal cells (hUcBdSCs) and umbilical cord blood–associated HIM. HSCs in cord From the Department of Hematology, Xinqiao Hospital, Third Military Medical University, Chongqing, China. Supported by grants from the National Natural Science Foundation (no. 30971109), Natural Science Foundation Project of Chongqing “CSTC” (no. 2009BA5011), and Innovation Foundation for Young Scientists of Third Military Medical University (no. 2009D226). Address reprint requests to Xing-Hua Chen, Department of Hematology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037, China. E-mail: [email protected]

© 2010 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710

0041-1345/–see front matter doi:10.1016/j.transproceed.2010.08.052

Transplantation Proceedings, 42, 3739 –3744 (2010)

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blood are more primitive than those in the bone marrow or blood, having the advantages of convenient collection, less GVHD after transplantation, high proliferative capacity, and long-term marrow reconstitution.18 –21 Our previous research has investigated the HIM.22 We previously showed that hUcBdSCs in human umbilical cord blood (hUCB), expand HSCs and enhance the formation of colony-forming units (CFUs) in vitro.23 The transplantation of human-derived stem cells into rodents with a normal immune system is the most common transplantation.21,24,25 In the present study, we extended our previous in vitro study23 to explore the role of hUcBdSCs in engraftment in haploidentical transplantation in mice. These studies may have potentially important clinical contributions for the development of new approaches for recipients of alloHSCT. MATERIALS AND METHODS Cell Separation Forty-five hUCB samples were collected from normal full-term deliveries at Xinqiao and Xi’nan Hospital, Chongqing, China. Blood collection was approved by the Institutional Ethics Committee in compliance with national guidelines regarding the use of fetal tissue for research purposes. Informed consent was obtained in all cases. No prisoners or materials from prisoners were used in the study. The volume of blood collected ranged from 55 to 140 mL. Cell separation was performed as described in our previous study.20 Briefly, the majority of red blood cells was depleted by a 6% gelatin sedimentation method. The leukocyte-rich fraction washed with Ca2⫹ and Mg2⫹-free phosphate-buffered saline solution (PBS) to remove the gelatin was loaded onto Percoll densitygradient fractionation columns (density 1.077 g/L; Pharmacia Biotech, Uppsala, Sweden). Cells were centrifuged at 400g for 20 minutes at 4°C. The mononuclear cells (MNCs) at the interface were washed with and resuspended in PBS.

CD34⫹ Cell Purification CD34⫹ cells were separated using a magnetic cell sorting system (Miltenyi Bioteo, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Briefly, 1 ⫻ 108 MNCs were mixed with anti-CD34 monoclonal antibody for 20 minutes at 4°C. Then caprine antimurine immunomagnetic beads were incubated with the MNCs for 15 minutes at 8°C. The percentage purity of the isolated positive fraction was determined using anti-CD34 fluorescein isothiocyanate (FITC) or phycoerythrin (PE) conjugated antibodies (Santa Cruz, CA, USA) with flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).

Establishment of hUcBdSCs The hUCB CD34⫹ cells were cultured in a Dexter system to obtain hUcBdSCs. CD34⫹ cells were resuspended in Dulbecco modified Eagle medium (DMEM; Gibco-Invitrogen, Carlsbad, Calif, USA) containing 12.5% fetal bovine serum (Hyclone, Logan, UT, USA), 12.5% horse serum (Gibco), 10⫺6 mmol/L hydrocortisone, 10 ng/mL recombinant human stem cell factor (Sigma, St Louis, Mo, USA), and 10 ng/mL recombinant human basic fibroblast growth factor (Sigma). Fresh medium was replaced after 48 hours, and then demidepopulated weekly accompanied by the addition of fresh medium. After the density of cells reached ⬎80% confluence,

ZHANG, CHEN, ZHANG ET AL the hUcBdSCs were subcultured (1:1) using the same medium and conditions.

Mice C57BL/6 (H-2b; termed B6) and F1 (H-2b/d; C57BL/6⫻BALB/c) male mice were purchased from the our animal center. The mice, 4 –12 weeks of age, were housed in a specific pathogen-free facility in microisolator cages. The experimental protocol was approved by our Animal Care Committee and was in agreement with “Guide for the Care and Use of Laboratory Animals,” published by the National Institutes of Health.

Haploidentical Transplantation With or Without hUcBdSCs Four- to 8-week-old inbred male black B6 mice were used as donors with 8 –12-week-old male F1 mice as bone marrow transplantation (BMT) recipients. They were maintained on water containing 250 mg/L erythromycin and 320 mg/L gentamicin to prevent infection with gram-positive and gram-negative bacteria, as well as an ad libitum diet after total body irradiation (TBI). The F1 mice were housed in ventilated cage racks after BMT. The mouse BMT model was established according to earlier reports by Li et al.26 Anesthetized recipient F1 mice were irradiated with 6-MV X-rays from a medical linear accelerator (7.0 Gy at 0.5 Gy/min) on the day before transplantation. On the day of transplantation, the donor was killed by cervical dislocation with aseptic removal of the femur and tibia to be kept on ice in DMEM supplemented with 0.5% fetal calf serum and 1% penicillinstreptomycin. Bones were opened at both ends to flush, bone marrow (BM) cells from the shaft by forcing cold DMEM through a 21-gauge needle inserted at the proximal end of the bone. Large fragments were removed by filtering the cell suspension through a 60-mm sterile nylon mesh. BM cells washed twice with PBS were resuspended in DMEM. The BM cells were counted, and viability determined by Trypan blue dye exclusion. 1 ⫻ 107 whole BM cells from B6 mice with or without 1 ⫻ 106 hUcBdSCs were injected intravenously into the lateral tail vein of irradiated F1 recipients; the control group was infused with physiologic saline solution. Acute GVHD was evident by rapid and sustained weight loss after recovery from irradiation, as well as from features such as hunchback, diarrhea, hair loss, and skin thickening.

Analysis of Chimerism Four weeks after transplantation, peripheral white blood cells (WBCs) were analyzed to determine the number of recipient- or donor-type cells by flow cytometry. Briefly, peripheral blood was incubated with PE-labeled antimouse H-2d and FITC-conjugated anti-H2b monoclonal antibodies, followed by hemolysis using BD PharM Lyse (BD Biosciences Pharmingen, San Jose, CA, USA). Stained cells were analyzed using FACScan (Becton Dickinson, Mountain View, CA, USA) with the percentage donor chimerism defined as donor/(donor ⫹ host) ⫻ 100%.

Statistical Analysis The results are expressed as mean ⫾ SEM. Statistical evaluation was performed using Student t tests with SPSS software (version 10.0). Survival curves were derived using Kaplan-Meier analysis, with mortality rates analyzed using a log-rank test. We used a significance level of P ⬍ .05.

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Table 1. Hematopoietic Reconstitution of White Blood Cells After Transplantation (ⴛ109/L) Days After Transplantation

BMT BMT ⫹ hUcBdSCs

1

3

5

7

10

14

21

5.03 ⫾ 0.40 5.73 ⫾ 0.32

0.40 ⫾ 0.10 0.87 ⫾ 0.15*

0.30 ⫾ 0.10 0.73 ⫾ 0.21*

0.47 ⫾ 0.15 1.07 ⫾ 0.15*

0.93 ⫾ 0.15 1.93 ⫾ 0.25*

1.63 ⫾ 0.25 2.33 ⫾ 0.21*

4.83 ⫾ 0.25 6.67 ⫾ 0.35*

The hemogram was monitored at 1, 3, 5, 7, 10, 14, and 21 days after transplantation. Peripheral blood samples were obtained by tail vein from 3 recipients in each experimental group, and full blood counts for each sample were determined using an automated hematology analyzer. The results showed that the decrease in the number of (WBCs) in the BMT with hUCBDSCs group was slower than in the BMT-only group, and hematopoietic reconstitution in the BMT with hUCBDSCs group was significantly faster than in the BMT only group (P values .077, .01, .03, .009, .004, .021, and .002, respectively). *P ⬍.05 versus BMT.

hUcBdSCs (3.7 ⫾ 0.82) versus BMT-alone group (7.2 ⫾ 1.14; P ⫽ .0000).

RESULTS Hematopoietic Reconstitution

The hemogram was monitored at 1, 3, 5, 7, 10, 14, and 21 days after transplantation. Peripheral blood samples were obtained from recipient tail veins, full blood counts for each sample were determined using an automated hematology analyzer. The decrease in the number of WBCs and platelets (PLTs) in the BMT with hUcBdSCs was slower than with BMT alone. The hematopoietic reconstitution in the BMT with hUcBdSCs group was significantly faster than in the BMT-alone group for WBC at 1, 3, 5, 7, 10, 14, and 21 days after transplantation, (P values .077, .01, .03, .009, .004, .021, and .002 respectively, for PLT at 1, 3, 5, 7, 10, 14, and 21 days after transplantation (P values .669, .04, .02, .01, .02, .001, and .002, respectively; Tables 1 and 2).

Donor Chimerism

At 28 days after transplantation the proportion of chimerism was 75.24 ⫾ 4.50% in the BMT with hUcBdSCs group, which was higher than the BMT-alone group (63.49 ⫾ 4.38%; P ⫽ .03). Fibroblast Assay

A Fibroblast assay was performed in both groups: 233.67 ⫾ 14.08 colony-forming units (CFU) per 106 MNCs were observed in the BMT with hUcBdSCs group versus 193.83 ⫾ 9.37 CFU per 106 MNCs in the BMT-alone group (P ⫽ .0002; Fig 2).

GVHD Observation

CFU Assay

The body weight of all recipient mice fell ⬃10%– 40% in the first week after TBI. In the BMT with hUcBdSCs group, the body weight began to increase ⬃2 weeks after transplantation, gradually increasing thereafter. In the BMTalone group, the change in body weight was similar to the change in body weight in the BMT with hUcBdSCs group. However, the body weight of mice in the BMT with hUcBdSCs group decreased significantly slower and increased faster than in the BMT-alone group (P ⫽ .01). Two of 20 mice died from acute GVHD in the BMT with hUcBdSCs group, with an average survival time of 28.15 ⫾ 1.28 days within 30 days after transplantation. Six of 20 mice died from acute GVHD after transplantation in the BMTalone group with an average survival time of 24.60 ⫾ 1.95 days within 30 days after transplantation (P ⫽ 0.09; Fig 1). However, the scoring of clinical GVHD grade at 28 days after transplantation was much lower in the BMT with

The quality of CFUs from the bone marrow of recipients was representative of the hematopoietic capacity. We detected the numbers of CFUs in the recipients after transplantation. The results showed that the difference in the numbers of granulocyte/monocyte, erythrocyte, and megakaryocyte CFUs in the cotransplanted animals versus BMT alone were statistically significant (P values .003, .004, and .003, respectively; Fig 3). Tracking of hUcBdSCs

Our results showed that hUcBdSCs accelerated hematopoietic reconstitution in mice with haploidentical bone marrow transplantations. However, we could not unequivocally confirm whether the injected hUcBdSCs moved to the bone marrow. hUcBdSCs marked with CM-DiI were detected at 3 and 21 days after transplantation by fluorescence micros-

Table 2. Hematopoietic Reconstitution of Platelets After Transplantation (ⴛ109/L) Days After Transplantation

BMT BMT ⫹ hUCBDSCs

1

3

5

7

10

14

21

635.67 ⫾ 18.90 645.00 ⫾ 29.55

160.33 ⫾ 3.21 167.33 ⫾ 2.52*

84.67 ⫾ 3.21 95.67 ⫾ 4.16*

52.00 ⫾ 2.65 64.33 ⫾ 3.79*

98.67 ⫾ 2.52 112.67 ⫾ 6.03*

154.67 ⫾ 3.79 176.00 ⫾ 3.00*

588.67 ⫾ 10.50 673.33 ⫾ 16.56*

The hemogram was monitored at 1, 3, 5, 7, 10, 14, and 21 days after transplantation. Peripheral blood samples were obtained by tail vein from 3 recipients in each groups, and full blood counts for each sample were determined using an automated hematology analyzer. The results showed that the decrease in the number of platelets in the BMT with hUCBDSCs group was slower than in the BMT-only group, and hematopoietic reconstitution in the BMT with hUCBDSCs group was significantly faster than in the BMT-only group (P values .669, .04, .02, .01, .02, .001, and .002, respectively). *P ⬍ .05 versus BMT.

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Fig 1. Kaplan-Meier survival curve in mouse haploidentical transplantation within 30 days after transplantation. Two of 20 mice died of acute GVHD in the BMT with hUCBDSCs group, with an average survival time of 28.15 ⫾ 1.28 days. Six of 20 mice died of acute GVHD in the BMT group, with an average survival time of 24.60 ⫾ 1.95 days. The difference in survival time was not statistically significant (P ⫽ .09).

copy (Leica DMIRB with Leica Qwin software, Germany) which indicated that hUcBdSCs indeedly moved to the bone marrow (Figure 4). DISCUSSION

The present study explored hematopoietic reconstitution by hUcBdSCs in haploidentical bone marrow transplantation

Fig 2. The numbers of fibroblast CFUs (CFU-F) with bone marrow MNCs at 21 days after transplantation in mouse haploidentical transplantation. The average numbers were 233.67 ⫾ 14.08/106 MNCs and 193.83 ⫾ 9.37/106 MNCs in the BMT with hUCBDSCs group and the BMT group, respectively; the difference was statistically significant (P ⫽ .0002).

ZHANG, CHEN, ZHANG ET AL

Fig 3. The numbers of CFUs with the bone marrow MNCs at 21 days after transplantation in HLA-haploidentical hematopoietic stem cell transplantation mice. The average numbers of granulocyte/monocyte (GM), erythrocyte (E), and megakaryocyte (Mg) CFUs in the BMT with hUCBDSCs group were 95.00 ⫾ 3.61, 474.33 ⫾ 7.02, and 170.00 ⫾ 8.19 and in the BMT group were 78.33 ⫾ 2.52, 443.00 ⫾ 6.00, and 137.00 ⫾ 3.61, respectively; the difference was statistically significant (P values .003, .004, and .003, respectively).

in mice. hUcBdSCs improved engraftment in transplanted mice, with infused hUcBdSCs moving to the bone marrow. T-cell– depleted allo-HSCT from an HLA-haploidentical relative is a feasible option for patients who need an allograft but lack an HLA-identical donor. However, the high rate of graft failure was mainly mediated by host alloreactive T cells that escaped the preparative regimen.27–29 Recipients of T-cell– depleted allo-HSCT from an HLA-disparate relative are also exposed to an increased risk of life-threatening infections, especially viral etiology, owing to the delay in hematopoiesis and adaptive immunity.28,30 Allo-HSCTs with T cells also have a risk of serious GVHD, infections, etc.31–33 Our previous study showed that hUcBdSCs effectively expand hematopoietic stem cells and enhance the formation of CFUs in vitro.23 The engraftment of stem cells is necessary to reconstitute posttransplantation hematopoiesis. Therefore, expansion protocols should be designed to not jeopardize engraftment potential. Designing protocols that allow true expansion of stem cells requires the support of hematopoiesis in vivo. Transplantation of HSCs into rodents has been the most common specific transplantation.21,24,25 However, it was unknown whether hUcBdSCs accelerated engraftment in haploidentical bone marrow transplantation recipients. The present results indicate that hUcBdSCs improved engraftment in haploidentical transplantation in mice. Complications, especially GVHD and infections, pose serious problems for HLA-haploidentical HSCT. After allo-HSCT, the recovery of immunity is mainly dependent on the HIM.33–37 Furthermore, HLA-haploidentical HSCT is an interesting alternative when no sibling or unrelated HLA-matched donor is available. Transplantation across

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fused hUcBdSCs migrated into bone marrow, which may repair the damaged HIM. Together, our data indicate that hUcBdSCs improved hematopoietic reconstitution in haploidentical transplantation in mice.

REFERENCES

Fig 4. Tracking of hUcBdSCs marked with CM-DiI in bone marrow in mouse haploidentical transplantation. Photos were taken by fluorescence microscopy (Leica DMIRB with Leica Qwin software, Germany). (A) hUcBdSCs were observed in bone marrow at 3 days after transplantation by smears; red fluorescence could be seen under the fluorescence microscope. (B) hUcBdSCs were observed in bone marrow at 21 days after transplantation by smears; red fluorescence could still be seen under a fluorescence microscope. Original magnification ⫻ 200/0.4).

the histocompatibility barrier is now possible. Interestingly, we observed that GVHD was reduced in haploidentical transplantation in mice using hUcBdSCs. In further trials we must assess the potential side effects of hUcBdSCs and their exact mechanisms to modulate GVHD or immunity. HSCs give rise to all types of blood cells, including lymphocytes and myeloid cells. In the bone marrow, the HSCs reside in the HIM. They are thought to supply essential factors required to maintain a pool of HSCs that ensure the appropriate numbers of mature blood cells throughout life.37,38 The present study show that the in-

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