Transplantation of mesenchymal stem cells to enhance engraftment of ...

Leukemia (2007) 21, 1733–1738 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu

ORIGINAL ARTICLE Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells K Le Blanc1,2, H Samuelsson2, B Gustafsson3, M Remberger2,4, B Sundberg2, J Arvidson5, P Ljungman1, H Lo¨nnies2, S Nava2 and O Ringdeń2,4 1

Hematology Center, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden; 2Division of Clinical Immunology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden; 3Department of Pediatrics, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden; 4Center for Allogeneic Stem Cell Transplantation, Karolinska University Hospital Huddinge, Stockholm, Sweden and 5Department of Women’s and Children’s Health, University Hospital, Uppsala, Sweden

Seven patients underwent treatment with mesenchymal stem cells (MSCs), together with allogeneic hematopoietic stem cell transplantation (HSCT). MSCs were given to three patients for graft failure and four patients were included in a pilot study. HSCT donors were three human leukocyte antigen (HLA)identical siblings, three unrelated donors and one cord blood unit. The conditioning was myeloablative in four patients and reduced in three patients. MSC donors were HLA-identical siblings in three cases and haploidentical in four cases. Neutrophil counts 40.5 109/l was reached at a median of 12 (range 10–28) days. Platelet counts 430 109/l was achieved at a median of 12 (8–36) days. Acute graft-versus-host disease (GVHD) grade 0–I was seen in five patients. Two patients developed grade II, which in one patient evolved into chronic GVHD. One severe combined immunodeficiency (SCID) patient died of aspergillosis, the others are alive and well. One patient, diagnosed with aplastic anemia had graft failure after her first transplantation and severe Henoch–Scho¨nlein Purpura (HSP). After retransplantation of MSCs and HSCs, she recovered from both the HSP and aplasia. Thus, co-transplantation of MSC resulted in fast engraftment of absolute neutrophil count (ANC) and platelets and 100% donor chimerism, even in three patients regrafted for graft failure/rejection. Leukemia (2007) 21, 1733–1738; doi:10.1038/sj.leu.2404777; published online 31 May 2007 Keywords: mesenchymal stem cells (MSCs); hematopoietic stem cell transplantation (HSCT); hematopoietic engraftment

engraftment.8,9 Both fetal and adult human MSCs promote hematopoietic engraftment in immunodeficient mice and fetal sheep.10–12 Reconstitution of myeloid, lymphoid and megacaryocytic lineages is supported by MSCs.11,13 Furthermore, MSCs have low immunogenicity and immunomodulatory effects through inhibition of T-cell proliferation to alloantigens and mitogens in vitro.14–16 In vivo, in humans, autologous and allogeneic MSCs are safe to infuse with no acute adverse events and no formation of ectopic tissue.17–20 The immunomodulatory effect in vivo is evident by prolongation of skin allograft survival and reversal of therapy-resistant acute graft-versus-host disease (GVHD).14,21,22 Thus, MSCs may be used to fasten hematopoietic three-lineage engraftment and to prevent rejection. We here report the use of allogeneic MSCs to enhance engraftment in a pilot study and to prevent rejection in three patients with previous graft failure/rejection. Four of the patients received human leukocyte antigen (HLA)-haploidentical MSCs. This is the first report on the use of haploidentical MSCs as cotransplantation for HSCT.

Materials and methods

Patients

Introduction Mesenchymal stem cells (MSCs) may be isolated from bone marrow, blood, adipose tissue, fetal tissue and cord blood.1–5 MSCs have the capacity to differentiate in vitro and in vivo into several mesenchymal tissues, such as bone, cartilage and fat.1,6,7 In addition, MSCs have been suggested to be the precursor cells in the bone marrow stroma that provide a scaffold and promote hematopoiesis. They have therefore been suggested to enhance engraftment after autologous and allogeneic hematopoietic stem cell transplantation (HSCT). After high-dose chemoradiotherapy before HSCT, the marrow stroma is damaged and this may affect and delay hematopoietic Correspondence: Professor K Le Blanc, Karolinska Institutet, Division of Clinical Immunology, Karolinska University Hospital Huddinge, F79, SE-141 86 Stockholm, Sweden. E-mail: [email protected] Received 6 March 2007; accepted 26 April 2007; published online 31 May 2007

Between December 2003 and December 2005, seven patients were co-transplanted with MSCs together with allogeneic MSCs. Follow-up was January 1, 2007. Three patients were diagnosed with leukemia, two with aplastic anemia and two with severe combined immunodeficiency (SCID) (Table 1). Three patients received MSCs to prevent graft rejection/failure, and four patients to enhance hematopoietic engraftment (Table 2). The study was approved by the Institutional Review Board and the Ethics Committee at Karolinska University Hospital Huddinge. Patients and MSC donors gave written informed consent.

Donors Allogeneic HSCT donors were HLA-identical siblings in three cases, HLA-A, -B and -DRb1 identical matched unrelated donors (MUD) in two, one major HLA antigen mismatched unrelated donor (MMUD) in one and an unrelated one HLA antigen mismatched cord blood in one (Table 1). HLA typing was performed by genomic high-resolution DNA-based typing (PCR-sequence-specific primer (PCR-SSP)).23 Peripheral blood stem cells (PBSCs) were mobilized with granulocyte-colony stimulating factor (G-CSF).24

MSCs to enhance engraftment of allogeneic HSCs K Le Blanc et al

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Conditioning Two patients with graft failure received fludarabine (Flu; 30 mg/ m2/day) for 3 days, combined with thymoglobulin (7.5–8 mg/kg; Genzyme, Waltham, MN, USA).25 One patient with acute myeloid leukemia (AML) and a boy with SCID were treated with busulfan (Bu; 16 mg/kg), followed by cyclophosphamide (Cy; 120 and 200 mg/kg, respectively).26 A patient with acute lymphoblastic leukemia (ALL) received Cy (120 mg/kg), combined with fractionated total body irradiation (TBI), 3 Gy for 4 days. A boy with severe aplastic anemia (SAA), who was regrafted due to a previous rejection, received Flu (30 mg/m2/day) for 5 days, Cy (60 mg/kg) and TBI (3 Gy/day) for 2 consecutive days.27 One girl with SCID received Flu (30 mg/m2/day) for 5 days, combined with melphalan (Mel; 140 mg/m2).28

Prophylaxis and treatment of GVHD Before HSCT, six patients were treated with thymoglobulin (6–8 mg/kg).25 Patient 4 developed an anaphylactoid reaction by thymoglobulin and received campath (cam; 10 mg) instead (Schering Nordic, Stockholm, Sweden). For GVHD prevention, methotrexate (Mtx) was combined with cyclosporine A (CsA) in three patients.26,27,29 Two patients were treated with CsA alone. Prednisolone (Pred) was used for the patient receiving a cord blood transplant instead of Mtx and one patient with SAA received tacrolimus (Fk) combined with sirolimus.30 Grade I acute GVHD was treated with Pred (2 mg/kg/day) according to our transplant protocol.31 Acute GVHD was graded from I to IV, according to accepted criteria.32 Supportive care was previously described in detail.31

matching of MSC donors and their respective patient is shown in Table 2. Heparinized bone marrow cells were separated on Redigrad (Amersham Biosciences AB, Uppsala, Sweden) as previously described in detail.19 The mononuclear cells were collected, washed and resuspended in Dulbecco’s modified Eagles medium-low glucose (DMEM-LG; Life Technologies, Gaithersburg, MD, USA), with a selected batch of 10% fetal bovine serum (National Veterinary Institute, Uppsala, Sweden). The cells were plated at 160 000 cells/cm2 at 371C in 5% CO2. When near confluence, the cells were detached by trypsin and ethylenediamine tetraacetic acid (EDTA; Invitrogen Corp., Grand Island, NY, USA), passaged and replated at 4000 cells/cm2. The cells were harvested at passages 2–3 (Table 2). MSCs were detached, washed in CliniMACS PBS-EDTA buffer (AmCell Myltenyi Biotech GmbH, Gladbach, Germany) and resuspended in NaCl with 10% AB plasma. Flow cytometry was performed using a FACSort and analyzed with CellQuest software (Becton Dickinson, San Jose, CA, USA). The MSCs expressed CD105 and CD73, but not CD34, CD45 or CD14. They were positive for HLA class I, but generally not for class II. The MSC suspensions were culture-negative for bacteria and mycoplasma before infusion. A cell dose of 1 106 MSC/kg was infused to each of the patients within 4 h of HSC infusion.

PCR for detection of donor and recipient chimera cells PCR amplification of variable number of tandem repeats was used to evaluate donor/recipient chimerism in CD3 þ , CD19 þ and CD33 þ cells.33

Results

Harvesting and expansion of MSCs

Safety

Around 50 ml of bone marrow was aspirated from each of the seven donors, median age was 31 (range 26–37) years. The Swedish Medical Product Agency approved the method. HLA-

MSCs were infused during 15 min through a central venous line. There was no acute toxicity and there were no signs of ectopic tissue after transplant.

Table 1 Patient UPN 1 2 3 4 5 6 7

Age/ Diagnosis SCT HSCT donor Match gender no. Age/gender

457 994 1020 1047 1110 1126 1143

44/M 34/F 38/M 9/M 12/F 1/M 2/F

CML CP2 AML CR2 ALL SAA SAA SCID SCID

3 1 1 2 2 1 1

34/M 35/F 35/F 36/F 27/M 0/F 36/M

Sib MUD Sib MMUD Sib MMUD MUD

Cell HSCT dose Conditioning source CD34 106/kg

GvH CMV serology prophylaxis Donor/recipient

PBSC PBSC PBSC BM BM CB PBSC

CsA CsA+MTX CsA+MTX Fk+Rapa CsA CsA+Pred CsA+MTX

20.8 7.2 8.3 2.65 3.39 0.21 68.9

Flu+ATG Bu+Cy+ATG FTBI+Cy FTBI+Cy+ATG+Cam Flu+ATG Bu+Cy+ATG Flu+Mel+ATG

7 7 7 7 +/+ / /

Abbreviations: ALL, acute lymphatic leukemia; AML, acute myeloic leukemia; ATG, antithymoglobulin; BM, bone marrow; Bu, busulphan; Cam, campath; CB, cord blood; CML, chronic myeloic leukemia; CMV, cytomegalovirus serology in donor and recipient; CsA, cyclosporine A; Cy, cyclophosphamide; F, female; Fk, Tacrolimus; Flu, fludarabine; FTBI, fractionated total body irradiation; GVHD, graft-versus-host disease; HSCT, hematopoietic stem cell transplantation; M, male; Match, donor–recipient HLA match; Mel, melphalan; MMUD, HLA mismatched unrelated donor; MTX, methotrexate; MUD, matched unrelated donor; PBSC, peripheral blood stem cells; Rapa, rapamycin; Sib, HLA identical sibling. Table 2

Indications for MSCs, donor HLA match, MSC dose and passage

Patient

UPN

MSC indication

1 2 3 4 5 6 7

457 994 1020 1047 1110 1126 1143

Graft failure Enhanced engraftment Enhanced engraftment Graft rejection Graft failure Enhanced engraftment Enhanced engraftment

MSC donor age/gender 35/M 26/F 35/F 37/F 27/M 31/M 31/M

MSC donor HLA match Sib Haplo Sib Haplo Sib Haplo Haplo

Cell dose 106/kg

Cell passage

1 1 1 1 1 1 1

2 2 2 3 2 3 3

Abbreviations: F, female; Haplo, HLA haploidentical related donor; M, male; MSC, mesenchymal stem cells; Sib, HLA identical sibling. Leukemia

Dead, aspergillosis, 8 months Alive and well, 10 months 0 0 7 0 6 1 2 2 45 11 36 11 1126 1143 6 7

24 11

1110 5

Abbreviations: ANC, absolute neutrophil count; EBV, Epstein–Barr Virus; RBC, red blood cells; VOD, veno occlusive disease of the liver. a Days after transplant.

I II

0 0

Fever of unknown origin VOD EBV viremia 0 2 2 31 24

0 0 0 3 1 3 6 0 0 12 15 32 11 12 20 12 12 28 994 1020 1047

16

0 0 + I I II

0 I

Elevated liver enzymes Hemorrhagic cystitis None None 0 1 2 11

Acute Platelets RBC Platelets 450 Platelets 430

8 10

2 3 4

Patient 1 with chronic myeloid leukemia in chronic phase received PBSCs from his HLA-identical sibling in 1994. In 1999, he developed graft failure. He received a booster marrow in 2000 without any effect and was, therefore, retransplanted with myeloablative conditioning using the same donor. In 2004, he again had poor hematopoiesis and bronchopneumonia. He was regrafted with PBSCs combined with MSCs from the same donor. He had an uneventful post-transplant course, apart from

ANC40.5

Outcome in patients undergoing retransplantation

UPN

All patients became 100% donor CD3, CD19 and CD33 chimeras within 30 days after transplantation.

Patient

Donor–recipient chimerism analysis

Engraftmenta ( 109/l)

Four patients developed localized skin rashes, which responded to steroids (Table 3). Two patients developed mild gastrointestinal GVHD, which responded to steroid therapy. Patient 4 developed chronic GVHD, which required long-term CsA and steroids. Eight months post transplant, he also developed listeria meningitis, which required ventilatory assistance. He had repeated bronchopneumonias, which responded to antibiotics. Furthermore, he also had three Varicella–Zoster infections, which responded to valciclovir. None of the other patients had any serious late infections.

Table 3

GVHD and late infections

Engraftment, transfusions, graft-versus-host disease (GVHD) and outcome

Owing to an infection at the central venous line catheter in patient 6 and spreading cellulitis, the catheter was replaced. This patient had septicemia by pseudomonas and coagulasenegative staphylococci and received seven granulocyte transfusions. Apart from this patient, none of the other patients had septicemia during the first month after HSCT.

Transfusions units

Early infections

Granulocyte

Toxicity

GVHD

Chronic

Patient 1, who received a third transplant, developed liver toxicity with rising liver enzymes, which responded to acetylcystein (100 mg/kg/day) (Table 3).34 Patient 2, a 34-yearold woman with AML in CR2, developed hemorrhagic cystitis on day 9, which resolved after intravenously (i.v.) hydration, oral cyklocapron for 3 days and early engraftment. Patient 5 had high fever of unknown origin, was treated with cephalosporine and developed an urticarial exanthema, slight hematuria and increased liver enzymes. After discontinuation of cephalosporine and conversion into imipenem, fever and all other parameters normalized. Patient 6 developed veno-occlusive disease of the liver (VOD), which responded to acetylcystein and defibrotide.34 Patient 7 had an increase in Epstein–Barr virus (EBV) DNA to 15 000 copies, fever but no clinical signs of EBV lymphoma. DNA copies decreased after two doses of Mabthera. Two patients had CMV reactivation analyzed by PCR, but no patient developed CMV disease.

457

Toxic side-effects

1735

1

Outcome post transplant

An absolute neutrophil count (ANC) 40.5 109/l was seen after 12 (10–28) days (Table 3). Platelets count 430 109/l was achieved after 12 (8–36) days and 450 109/l after 15 (11–45) days. During the pancytopenic phase and the first month after transplant, the patients received 2 (0–6) erythrocyte transfusions. A median of 2 (1–6) platelet transfusions was given.

Alive and well, 19 months

Engraftment and transfusions

Alive and well, 37 months Alive and well, 32 months Chronic GVHD of skin and gut, repeated bacterial infections, 28 months Alive and well, 15 months


Leukemia


1736 increased liver enzymes. He now has normal hematopoiesis almost 2 years after his third transplant. Patient 4, a 9-year-old boy was diagnosed with SAA in 2003. He underwent HSCT with an unrelated donor in February 2004. He rejected the graft and was regrafted with a second unrelated donor together with MSCs from his haploidentical mother in September 2004. His early post-transplant course was uncomplicated. Nine months after transplant, he developed gastrointestinal GVHD that responded to steroids, but subsequently, he developed chronic GVHD of the skin and experienced repeated infections. He continues to manage well on Fk and low-dose steroids. His Karnofsky score is 80%. Patient 5, a 12-year-old girl with SAA diagnosed in 2000, received bone marrow from her HLA-identical brother in May 2000. She had pancytopenia and received a booster graft from the same donor after 1 year. Chimerism showed 100% donor cells, but pancytopenia continued. In 2003, she developed Henoch-Scho¨nlein vasculitis and required immunosuppression with CsA. She had petechia and exanthema on legs and arms. Owing to graft failure, she was admitted for retransplantation with HSCs and MSCs from the same donor. Apart from a short episode of fever of unknown origin, she had an uneventful posttransplant course with prompt engraftment and only required a few transfusions. Her Henoch-Scho¨nlein purpura has completely resolved. At the last follow-up, she was feeling well, had a hemoglobin value of 140 g/l, a neutrophil count of 1 109/l and a platelet count of 60 109/l.

Discussion As in previously reported studies, there was no acute toxicity related to the MSC infusion in our patients.17,18,21,22 Even haploidentical MSCs are safe to infuse. There was no sign of ectopic tissue formation upon clinical examination during a follow-up of 3 years, or upon autopsy in the diseased patient. Computer tomography was only performed when otherwise clinically indicated, to limit the patients’ exposure to radiation. One indication for MSCs at the time of transplant was to enhance engraftment, since chemoradiotherapy damages the marrow stroma.8,9,35–37 Indeed, ANC engraftment with 40.5 109/l on median day 12 with no G-CSF support is fast and compares favorably to the previous experience. Using HSCT with HLA identical sibling donors or MUD, time to ANC 40.5 109/l was a median of 19–20 days.31 With a reduced intensity conditioning and PBSC, median time to ANC 40.5 109/l was 17 days in our unit.38 Even more impressive is a median of 12 days to reach platelet counts 430 109/l. Compared to ANC engraftment, platelet engraftment is often delayed after allogeneic HSCT. It should also be kept in mind that three of these patients had graft failure and were transfusion-dependent at the time of transplant. In accordance with a fast engraftment, transfusion requirements were also low with a median of two erythrocyte and platelet transfusions, respectively, in each patient. Small children generally do not require many transfusions, but for the adults in this study, the low requirement for transfusions is promising. The mechanisms whereby MSCs may enhance hematopoietic recovery remains unclear. After HSCT, stromal progenitors are predominantly recipient in origin.39 After co-transplantation with ex vivo expanded MSCs, a small subset of patients had low levels of donor MSCs in post-transplant MSC cultures more than 1 year post transplant.20,40 A low level of engraftment was observed in bone marrow biopsies but not in aspirates after infusion of haploidentical-related MSCs to a patient with severe Leukemia

aplastic anemia.41 In patients treated with third party-derived HLA-mismatched MSCs for steroid-resistant GVHD, MSC-donor HLA could be detected in organs affected by the GVHD but not in healthy tissue.22 Despite the limited capacity of MSCs to reconstitute the marrow microenvironment, MSCs promote engraftment of unrelated and umbilical cord-derived HSCs in nonobese-diabetic/severe compromised immune deficiency mice and fetal sheep.10–12 The enhancing effect is most prominent when the dose of hematopoietic cells is limited. It is possible that the release of cytokines in the circulation promotes either homing or proliferation of HSCs and may not require homing of MSCs to the bone marrow. A reduced transfusion requirement in co-transplanted patients will be further accessed in prospective randomized studies recently started in Europe, where recipients of unrelated bone marrow receiving myeloablative conditioning will be randomized in a double-blind fashion to co-transplantation with MSCs or not. Such a study will more exactly pinpoint the possible advantages on engraftment and transfusions using MSCs. All patients were 100% donor chimera within 1 month for CD3, CD19 and CD33, which may suggest that MSCs have affected donor cell engraftment and may have prevented rejection. Despite the prompt donor cell engraftment, none of the patients developed severe acute GVHD. There is a strong correlation between donor cell chimerism and severe acute GVHD.42 The absence of severe GVHD in these patients may in part result from the immunomodulatory effect by MSCs previously described in vitro and in vivo.14–16,19,21,22 Since MSCs may repair damaged tissue and preferentially home to sites of tissue injury, it was hoped that MSCs given at the time of HSCT may protect or prevent toxic side effects to chemoradiotherapy.43,44 However, protection of toxicity is not evident from the experience in this small series, where one patient developed VOD, another patient had liver toxicity with elevated liver enzymes and two patients experienced mild-tomoderate hemorrhagic cystitis. The ongoing prospective randomized studies may tell if MSCs protect from conditioning-related toxicity or not. It is possible that after transplantation, at the time of toxicity, the MSCs have either disappeared or been widely distributed in the body. After infusion, it has been difficult to detect MSCs, even using sensitive PCR techniques.22,45 There were but few infections in these patients. By fast engraftment of ANC, MSCs may prevent early bacterial and fungal infections. By modulating and preventing severe GVHD, MSCs may also prevent late infections and viral reactivation. During the pancytopenic phase, only one patient had septicemia, a boy with SCID (patient 6). However, this patient was immunocompromised since birth with repeated severe infections. Only two patients had CMV reactivation, despite that five were CMV-seropositive at the time of transplant. Four of them also had CMV-seronegative donors, which may increase the risk for CMV infection and disease post-transplant.46 None of the patients experienced CMV disease. The prospective randomized studies may shed some light on whether MSCs can fasten immune recovery and decrease the probability of post-transplant infections. Co-transplantation of MSCs and HSCs reconstituted hematopoiesis in the two patients with graft failure who did not respond to booster stem cells (patients 1 and 5). Also in patient 4 who rejected a previous graft, MSCs may have paved the way for the second transplant by immunomodulation and enhancement of donor hematopoiesis. These encouraging data will prompt us to treat all retransplantations due to rejection with MSCs. It is difficult to do a prospective randomized study, because retransplantation is a rather rare event and it will take many


1737 years before this can be evaluated in a prospective multicentered trial. Although this is a small pilot study with patients of different ages, diagnoses, disease stages, conditioning and age, it suggests that MSCs may have positive effects This will be further evaluated in a recently started randomized study. In conclusion, MSCs hold some promise to enhance hematopoietic engraftment, and donor chimerism with subsequent low probability of severe acute GVHD and infections.

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Acknowledgements We thank Inger Hammarberg and Kristina Gynning-Holmstro¨m for secretarial assistance and Maivor Ho¨glund-Lindecrantz for help with data collection. We also thank the staff at Center for Allogeneic Stem Cell Transplantation, Department of Pediatrics and Hematology, for compassionate and competent patient care. This study was supported by grants from the Swedish Cancer Society (0070-B02-16XAC, 4562-B02-02XBB, 4562-B05-05XCC), the Children’s Cancer Foundation (2000/067, 01/039, 05/007), the Swedish Research Council (K2003-32X-05971-23A, K200332XD-14716-01A, K2006-32X-14716-04-1, K2005-32P-1545701A), the Cancer Society in Stockholm, the Cancer and Allergy Foundation, the Tobias Foundation, the Swedish Society of Medicine, the Stockholm County Council, the Sven and EbbaChristina Hagbergs Foundation and Karolinska Institutet.

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