Human Application of Ex Vivo Expanded Umbilical Cord-Derived Mesenchymal Stem Cells: Enhance Hematopoiesis after Cord Blood Transplantation

Abstract

Delayed hematopoietic reconstitution after cord blood (CB) transplantation (CBT) needs to be overcome. Bone marrow-derived mesenchymal stem cells (BMMSCs) have been found to enhance engraftment after hematopoietic stem cell transplantation. However, getting BMMSCs involves an invasive procedure. In this study, umbilical cord-derived mesenchymal stem cells (UCMSCs) were isolated from Wharton's jelly and cryopreserved in the UCMSCs bank. Compared with BMMSCs, we found that UCMSCs had superior proliferative potential. We found that NOD/SCID mice cotransplanted with CB and UCMSCs demonstrated significant human CD45⁺ cell engraftment compared with those transplanted with CB alone. Then, 20 patients with high-risk leukemia were prospectively randomized to either receive cotransplantation of CB and ex vivo expanded banked UCMSCs or to receive CBT alone. No serious adverse events were observed in the patients receiving UCMSC infusion. The time to undergo neutrophil engraftment and platelet engraftment was significantly shorter in the eight patients receiving cotransplantation than that in the 12 patients receiving CBT alone (p = 0.003 and p = 0.004, respectively). Thus, application of ex vivo expanded banked UCMSCs in humans appears to be feasible and safe. UCMSCs can enhance engraftment after CBT, but further studies are warranted.

Keywords

Mesenchymal stem cells (MSCs)Umbilical cord Cord blood transplantation (CBT)

Introduction

Mesenchymal stem cells (MSCs) can have clinical applications in regenerative medicine and tissue repair in adverse health events (9) such as heart diseases (14), neurological problems (13), lung diseases, liver diseases, renal diseases (5), diabetes, and bone problems. The immunomodulatory properties of MSCs make them clinically valuable in immune disorders (8,15,25,31,35). In most previous reports, MSCs in human clinical applications were derived from bone marrow (BM) (4,16,18–22,27,29). However, harvesting BM-derived MSCs (BMMSCs) involves an invasive and painful procedure. MSCs also can be isolated without an invasive procedure from fetal tissues, such as amniotic fluid (2), amniotic membrane (23), placenta, cord blood (CB) (5), and umbilical cord (UC) (12,32). The UC is rich with MSCs, and UC-derived MSCs (UCMSCs) have been shown to be easy to isolate and culture (32). Therefore, the UC can be considered as an alternative source of MSCs for clinical applications.

Hematopoietic stem cell transplantation (HSCT) is an effective therapeutic modality for a variety of disorders. CB is a source of hematopoietic stem cells (HSCs), and there are some advantages in CB transplantation (CBT), including fast availability, tolerance of one to two human leukocyte antigen (HLA) mismatch, low incidence and severity of acute graft-versus-host disease (GVHD), low risk of transmitting infections, and lack of risks for donors (11). However, because of the limited number of HSCs in CB, patients receiving CBT have been found to have delayed hematopoietic engraftment, which might result in more complications, such as infections and hemorrhage, and prolonged hospitalization (11,30).

Previous studies have documented that cotransplantation of HSCs and BMMSCs can enhance hematopoiesis, reduce the incidence of GVHD, accelerate lymphocyte recovery, and reduce the risk of graft failure (4,16,18,21,22,27). BMMSCs were also reported to promote hematopoietic engraftment after CBT (22). Our previous studies found that no severe adverse effects were noted in patients receiving UCMSCs. We also found that UCMSCs could enhance engraftment after HSCT in patients with severe aplastic anemia and effectively treat severe acute GVHD (7,34). In the present study, after ex vivo and animal studies, we prospectively randomized subjects to investigate the safety and effect of cotransplantation of CB and UCMSCs in patients with high-risk leukemia.

Patients and Methods

Isolation and Culture of BMMSCs

BM cells of healthy individuals were obtained from iliac crest aspirates. The institutional review board (IRB) of the China Medical University Hospital approved this protocol, and written informed consents were obtained. Mononuclear cells were isolated from BM aspirates by Ficoll-Paque density centrifugation (1.077 g/ml; Amersham Biosciences, Uppsala, Sweden) and then seeded in minimum essential medium with a modification (α-MEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) and penicillin– streptomycin (Invitrogen). Cells were incubated at 37°C with 5% CO₂ in a humidified atmosphere. After 48 h, the medium with suspension of nonadherent cells was discarded, and fresh medium was added. Thereafter, the medium was replaced twice a week. When reaching 80–90% confluence, cells were detached with 0.25% trypsin-EDTA (Invitrogen) and replated at a concentration of 8.5 × 10³/cm² in 10-cm dishes.

Collection and Cryopreservation of UCMSCs

IRB approval and informed consent was obtained from all eligible UC donors who tested negative for human immunodeficiency virus (HIV)-I, HIV-II, human T-lymphotropic virus (HTLV)-I, HTLV-II, hepatitis B and C, cytomegalovirus (CMV), and syphilis and were free of any active infections during UCMSC harvesting. The UC was collected immediately after parturition and express-shipped to the UCMSC bank for MSC processing. UCMSCs were harvested from Wharton's jelly as previously described (7,34). Briefly, the main vessels were carefully removed, and the UC was sectioned to acquire Wharton's jelly. The jelly was digested using 1 mg/ml collagenase (Sigma, St. Louis, MO, USA) and plated in media (as above) at 5% CO₂ and 37°C in a humidified atmosphere. Forty-eight hours later, the suspended nonadherent cell-containing medium was discarded and replaced with fresh medium. The cells were detached with 0.25% trypsin-EDTA at 80–90% confluence and subcultured for further expansion. Once the cells had been proven negative for bacteria, fungi, mycoplasma, and endotoxins and UCMSCs were identified, the cells were cryopreserved in 10% dimethyl sulfoxide (DMSO; WAKChemie Medical GmbH, Steinbach, Germany) using a controlled-rate freezer and maintained in liquid nitrogen at the UCMSCs bank. All procedures for UCMSC processing complied with the current good tissue practice (GTP) requirements.

Identification of MSCs

BMMSCs and UCMSCs were immunolabeled with the following mouse anti-human antibodies: cluster of differentiation 14 (CD14), CD34, CD45, CD13, CD29, CD31, CD44, CD90, and HLA-DR (BD Biosciences, San Jose, CA, USA); CD105 (AbD Serotec, Oxford, UK); and CD73 (BD Pharmingen, San Diego, CA, USA). The cells were incubated with a secondary antibody, antimouse IgG-fluorescein isothiocyanate (FITC) or IgG-phycoerythrin (PE) (BD Biosciences) and analyzed by flow cytometry (BD Biosciences). The MSCs then underwent osteogenic, adipogenic, and chondrogenic differentiation under specific induction conditions (6,26).

Proliferative Potential Assay

The population doublings of the cultured UCMSCs (n = 10) and BMMSCs (n = 10) were calculated according to the equation: population doubling = log₂ (the number of viable cells at harvest/the number of seeded cells) (6). The population doubling time was derived from the time interval divided by the cumulative population doubling from passages 2 to 6.

Cotransplantation of NOD/SCID Mice with CB and UCMSCs

The male nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were obtained from the National Science Council (Taipei, Taiwan) and maintained at the Animal Center of China Medical University. There were two groups of 10 mice each (age 6–8 weeks) sublethally irradiated with 350 cGy (Varian, Palo Alto, CA, USA) and then transplanted with the following cells: (a) 10⁶ total nucleated cells (TNCs) in the CB together with 10⁶ UCMSCs and (b) 10⁶ TNCs in the CB alone. The cells in these two groups were suspended in 250 ml of phosphate-buffered saline (Invitrogen) and injected into the lateral tail veins. These mice were sacrificed 6 weeks after transplantation. Peripheral blood samples were collected by cardiac puncture, and BM was harvested by flushing femurs and tibias with phosphate-buffered saline. To document hematopoietic cells engraftment, these cells from peripheral blood and BM were stained with a FITC-conjugated antibody to human CD45 (BD Pharmingen), and the percentages of human CD45⁺ cells were measured by flow cytometry.

In addition, in order to compare the time of achieving complete chimerism between NOD/SCID mice cotransplanted with CB and UCMSCs and those transplanted with CB alone, we analyzed the percentages of human CD45⁺ cells in the peripheral blood collected from the tail vein every week in another two groups of 10 mice each (age 6–8 weeks) sublethally irradiated with 350 cGy and then transplanted. We defined the end point when complete chimerism (100% human CD45⁺ cells in peripheral blood) in mice was achieved.

Patients

After the IRB at the China Medical University Hospital and the Department of Health in Taiwan approved this study, children with high-risk leukemia but lacking HLA-matched siblings or unrelated donors were enrolled in this study at the China Medical University Hospital. The high-risk acute lymphoblastic leukemia (ALL) was defined as ALL with cytogenetic abnormalities indicating poor prognosis or in second remission. The high-risk acute myeloid leukemia (AML) was defined as AML with cytogenetic abnormalities indicating poor prognosis, in second remission, or with myelodysplastic syndrome. We prospectively randomized patients to receive cotransplantation of CB and UCMSCs or CBT alone. Parents or legal guardians of patients provided written informed consent for inclusion in this study. Written informed consent in accordance with the Declaration of Helsinki was also obtained from patients receiving UCMSC infusion by an independent physician to explain risks associated with UCMSC infusion. Patients who received CBT alone were enrolled as the control group.

Expansion, Thawing, and Infusion of UCMSCs

After we recruited suitable patients for the study (see Table 1 for demographics), we selected a unit of banked UCMSCs matching HLA based on antigen-level HLA-A, HLA-B, and HLA-DR typing with the recipients. UCMSC units and recipients should match at least three HLAs. The selected UCMSCs were thawed and subcultured in α-MEM supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO₂. The medium was replaced twice a week. When reaching 80–90% confluence, cells were detached with 0.25% trypsin-EDTA and replated. When adequate cell dosages were obtained, the expanded UCMSCs were then cryopreserved again. After UCMSC expansion and before recryopreservation, karyotyping and screening for bacteria, fungi, mycoplasma, and endotoxins tests were performed. After the cells were shown to have a normal karyotype and were negative for infectious contamination, these cells were shipped from the UCMSC bank to our hospital. On the day of CBT, the UCMSCs were thawed, washed (16), and intravenously infused into the recipient through a central venous line, followed by CB infusion 4 h afterward. Some residual UCMSCs and suspension were tested again to rule out infectious contamination. All cotransplanted patients received one unit of CB and one unit of expanded UCMSCs. The collection, cryopreservation, expansion, thawing, and infusion of UCMSCs are summarized in Figure 1.

Figure 1.

The flow chart of collection, cryopreservation, expansion, thawing, and infusion of umbilical cord-derived mesenchymal stem cells (UCMSCs). HLA, human leukocyte antigen.

Table 1.

Characteristics of Patients Receiving Cotransplantation of CB and UCMSCs

	Patient No.
	1	2	3	4	5	6	7	8
Age at transplant (years)	3.2	11.7	4.4	9.2	12.1	6.7	10.3	10.7
Gender	F	M	F	M	M	M	F	M
Diagnosis	AML in 2nd remission	AML with MDS	AML with MDS	ALL with Ph	AML with monosomy 7	AML in 2nd remission	ALL with Ph	ALL in 2nd remission
Characteristics of CB
HLA match	4/6	4/6	4/6	4/6	4/6	5/6	4/6	5/6
TNC dose (x10⁷/kg)	7.34	3.71	6.36	10.56	6.58	5.98	7.01	8.15
CD34⁺ cell dose (x10⁵/kg)	3.21	2.89	2.92	6.11	2.95	3.51	2.59	4.11
Gender of CB donor	F	M	M	M	M	F	F	M
Characteristics of UCMSCs
HLA match	5/6	4/6	3/6	4/6	5/6	3/6	4/6	4/6
UCMSC dose (x10⁶/kg)	3.38	5.76	9.41	10.12	2.44	5.66	8.92	8.61
UCMSC passage	3	3	4	4	2	3	4	3
Duration of expansion to sufficient UCMSCs (days)	6	7	8	8	5	6	7	7
Gender of UCMSCs donor	F	F	F	M	M	F	F	M
Engraftment
ANC>.5 × 10⁹/L(days)	16	15	9	12	13	10	8	11
Platelet count> 20 × 10⁹/L (days)	35	41	20	29	45	31	25	21
GVHD
Acute GVHD	None	Grade I	None	None	Grade II	None	Grade I	Grade I
Chronic GVHD	None	None	None	None	Limited	None	None	None
Outcome/Follow-up (months)	Alive, leukemia in complete remission/11	Alive, leukemia in complete remission/15	Alive, leukemia in complete remission/25	Alive, leukemia in complete remission/27	Expired due to leukemia relapse/8	Alive, leukemia in complete remission/14	Alive, leukemia in complete remission/18	Expired due to leukemia relapse/21

ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ANC, absolute neutrophil count; CB, cord blood; F, female; GVHD, graft-versus-host disease; HLA, human leukocyte antigen; M, male; MDS, myelodysplastic syndrome; Ph, Philadelphia chromosome; TNCs, total nucleated cells; UCMSCs, umbilical cord-derived mesenchymal stem cells.

Toxicity Assessment About UCMSCs

Vital signs were monitored at 15 and 30 min, 1, 2, 4, 6, 10, and 14 h after UCMSC infusion. Patients were monitored continuously for UCMSC infusion-related toxicities defined as alterations in vital organ functions, which cannot be explained by other intercurrent complications such as infection or chemoradiotherapy within 28 days of UCMSC infusion. To assess the presence of ectopic tissue formation, patients were evaluated by magnetic resonance imaging (MRI; GE Medical Systems, Milwaukee, WI, USA) and positron emission tomography (PET; GE Medical Systems) every 3 months after UCMSC infusion.

CB Selection

Searches for unrelated CB donors were processed through the CB bank approved by the Department of Health in Taiwan. The compatibility of HLA-A, -B, and -DRB1 was assessed by high-resolution typing. The CB units were selected according to the TNCs in the CB units and the HLA match between recipients and the CB units. The selected CB units had to contain at least 3 × 10⁷ TNCs/ kg of the recipients (prethaw). A minimum of four HLA antigens shared with the recipients was required, and only one HLA-DRB1 single-locus mismatch was allowed. All patients enrolled in this study received one unit of CB, whether cotransplanted with UCMSCs or transplanted alone. Any patient receiving two or more units of CB was excluded.

Conditioning Regimens, GVHD Prophylaxis, and Supportive Care

Conditioning regimens were different according to ALL or AML. Patients with ALL received cyclophosphamide (Baxter Oncology GmbH, Halle, Germany) 60 mg/kg/day intravenously for 2 days, etoposide (Bristol-Myers Squibb, Princeton, NJ, USA) 30 mg/kg intravenously for 1 day, and total body irradiation 200 cGy twice a day for 3 days. Patients with AML were treated with intravenous busulfan (Otsuka, Tokyo, Japan) 1 mg/kg intravenously every 6 h for 4 days, and cyclophosphamide 60 mg/kg/day intravenously for 2 days. GVHD prevention consisted of intravenous or oral cyclosporine (Novartis, Basle, Switzerland) and intravenous methylprednisolone (Pfizer, NY, USA). All patients received rabbit thymoglobulin (Genzyme, Marcy L'Etoile, France) 2.5 mg/kg/day intravenously for 4 days. Patients received antimicrobial prophylaxis with oral ciprofloxacin (Bayer, Leverkusen, Germany), oral voriconazole (Pfizer, Illertissen, Germany), oral trimethoprim-sulfamethoxazole (Shionogi, Osaka, Japan), and intravenous acyclovir (GlaxoSmithKline, Torrile, Italy). CMV pp65 antigen and pp65 genes were tested from peripheral blood samples every week for CMV infection (24). Within 4 h of specimen collection, the CMV pp65 antigen assay was carried out using the Light Diagnostics™ CMV pp65 Antigenemia Immunofluorescence Assay (Chemicon International, Temecula, CA, USA) according to the manufacturer's instructions. The result was expressed as the number of CMV antigen-positive cells per 2 × 10⁵ leukocytes in the slide. For detection of CMV pp65 genes, total nucleic acids were extracted from 350 μl of EDTA-anticoagulant whole blood and eluted in 200 μl of elution buffer using the automatic Bio Robot Ez1 extractor (Qiagen, Valencia, CA, USA). According to the manufacturer's protocol, CMV DNA was quantified using a commercially available real-time PCR test (TaqMan Q-CMV Rt Complete KIT; Cepheid, Torino, Italy). Once CMV was detected, ganciclovir (Roche, Basel, Switzerland) was administered intravenously. After transplantation, complete blood count was checked every day, and granulocyte colony-stimulating factor (G-CSF; Kirin, Tokyo, Japan; 10 μg/kg/day) was administrated subcutaneously until absolute neutrophil count (ANC) > 1.0 × 10⁹/L. The ANC is calculated by multiplying the total leukocyte count by the percentage of segmented neutrophils and bands. Short tandem repeat analysis was used for assessment of engraftment after CBT (17). For short tandem repeat analysis, DNA was prepared from mononuclear cells obtained from the CB and peripheral blood of the recipient before CBT. Specific primers designed to flank repeated units of the apolipoprotein B (APO-B) and D1S80 gene regions were used for PCR amplification. Transplant-related mortality was defined as patients expired due to CBT.

Statistics

A boxplot was made to compare the human CD45⁺ cells between NOD/SCID mice cotransplanted with CB and UCMSCs and those transplanted with CB alone. A Mann–Whitney test and Fisher's exact probability test were used to assess differences between patients receiving cotransplantation of CB and UCMSCs and patients receiving CBT alone. The data are presented as median and range. A value of p < 0.05 was considered significant.

Results

Comparisons Between UCMSCs and BMMSCs

UCMSCs showed the same uniform spindle-shaped morphology as BMMSCs, and they were both positive for CD13, CD29, CD44, CD73, CD90, and CD105 but negative for CD14, CD31, CD34, CD45, and HLA-DR. Under specific induction conditions, UCMSCs and BMMSCs possessed the capacity for adipogenic, osteogenic, and chondrogenic differentiation (6,26). A significant increase in cumulative population doubling from passages 2 to 6 was observed between UCMSCs (median, 6.3; range, 4.6–7.8) and BMMSCs (median, 2.9; range, 1.6–4.4) (p = 0.004). The population doubling time of UCMSCs (median, 1.4 days; range, 1.1–1.9 days) was significantly shorter than that of BMMSCs (median, 9.6 days; range, 11.7–7.2 days) (p = 0.003).

Comparisons Between NOD/SCID Mice Cotransplanted with CB and UCMSCs and Those Transplanted with CB Alone

As shown in Figure 2, we found that NOD/SCID mice transplanted with 10⁶ TNCs in the CB together with 10⁶ UCMSCs demonstrated a significantly higher median 28.2% (range, 24.6–33.1%) number of human CD45⁺ cells engrafting in the peripheral blood than those transplanted with 10⁶ TNCs in the CB alone (median, 5.3%; range, 4.2–6.5%; human CD45⁺ cell engraftment in the peripheral blood) (p = 0.001). NOD/SCID mice transplanted with 10⁶ TNCs in the CB together with 10⁶ UCMSCs demonstrated a significantly greater median 6.9% (range, 5.9–7.3%) number of human CD45⁺ cells engrafting in the BM than those transplanted with 10⁶ TNCs in the CB alone (median, 1.7%; range, 1.5–2.3%; human CD45⁺ cell engraftment in the BM) (p = 0.015).

Figure 2.

Cotransplantation of umbilical cord-derived mesenchymal stem cells (UCMSCs) enhanced engraftment of human CD45⁺ cells in NOD/SCID mice. The nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice that were transplanted with cord blood (CB) together with 10⁶ UCMSCs demonstrated a significantly higher human CD45⁺ cell engraftment in the peripheral blood and bone marrow compared to those transplanted with CB alone.

Of the 10 mice receiving cotransplantation of CB and UCMSCs, complete chimerism was observed after a median time of 9 weeks (range, 8–11 weeks). Of the 10 mice receiving CBT alone, complete chimerism was not observed until after a median time of 16 weeks (range, 14–20 weeks). By defining complete chimerism in mice as the end point, we found that faster engraftment was achieved in mice receiving cotransplantion with CB and UCMSCs in comparison to those receiving CBT alone (p = 0.007). Based on the results of animal experimental investigation, we found that the cotransplantation of UCMSCs did enhance engraftment of CBT.

Characteristics of Patients and CB

The characteristics of eight patients with a median age of 9.8 years (range, 3.2–12.1 years) receiving cotransplantation of CB and UCMSCs are summarized in Table 1. Of the 8 units of CB cotransplanted with UCMSCs, three CB donors were female, and five were male. Of the eight patients, three patients had ALL, and five patients had AML. Twelve patients with a median age of 8.5 years (range, 3.6–13.1 years) received CBT alone. Of these 12 patients, five patients had ALL, and seven patients had AML. The characteristics of the 12 patients are summarized in Table 2. The age at transplant, gender, leukemia type, TNCs in CB, and CD34 cells in CB between patients receiving cotransplantation and those receiving CBT alone were not significantly different (Table 3).

Table 2.

Characteristics of Patients Receiving CBT Alone

				Characteristics of CB				Engraftment
Patient No.	Age at Transplant (Years)	Gender	Diagnosis		HLA Match	TNC Dose (×10⁷/kg)	CD34⁺ Cell Dose (×10⁵/kg)	Gender of CB Donor	ANC>0.5 × 10⁹/L (Days)	Platelet Count>20 × 10⁹/L
(Days)	Acute GVHD	Chronic GVHD	Outcome/Follow-up (Months)
1	5.4	M	ALL in 2nd remission	5/6	6.12	3.51	M	35	115	None	None	Expired due to leukemia
2	9.8	M	AML with MDS	4/6	6.69	3.48	M	36	126	Grade I	Limited	relapse/13 Alive, leukemia
3	3.6	F	ALL in 2nd remission	4/6	9.18	8.12	F	18	59	Grade I	None	in complete remission/13 Alive, leukemia in complete remission/12
4	4.7	M	ALL with Ph	5/6	8.11	6.11	F	19	62	None	None	Alive, leukemia in complete remission/15
5	9.1	M	AML in 2nd remission	4/6	7.34	3.60	M	22	84	None	None	Expired due to leukemia
6	11.1	F	AML in 1st	5/6	7.01	3.41	F	30	110	None	None	relapse/10 Alive, leukemia
7	10.2	F	remission ALL with Ph	4/6	7.08	4.2	F	20	55	Grade II	Limited	in complete remission/21 Alive, leukemia
8	12.5	M	ALL in 2nd remission	5/6	3.52	3.16	F	43	95	Grade I	None	in complete remission/16 Expired due to Pseudomonas
9	7.2	M	AML with MDS	4/6	11.25	10.90	M	19	47	Grade II	Limited	sepsis/2 Alive, leukemia
10	13.1	F	AML in 1st	4/6	5.14	2.80	F	41	135	Grade III	Extensive	in complete remission/31 Expired due
11	6.1	F	remission AML in 2nd	5/6	11.51	8.91	M	20	42	Grade I	None	to pulmonary aspergillosis/9 Alive, leukemia
12	7.9	F	remission AML in 2nd remission	5/6	10.11	5.62	F	17	49	Grade I	Limited	in complete remission/26 Alive, leukemia in complete remission/29

ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ANC, absolute neutrophil count; CB, cord blood; CBT, cord blood transplantation; F, female; GVHD, graft-versus-host disease; HLA, human leukocyte antigen; M, male; MDS, myelodysplastic syndrome; Ph, Philadelphia chromosome; TNCs, total nucleated cells.

Table 3.

Comparison Between Patients Cotransplanted With CB and UCMSCs and Those Transplanted With CB Alone

	Patients Receiving Cotransplantation	Patients Receiving CBT Alone	p Value
No.	8	12
Age at transplant (years), median (range)	9.8 (3.2–12.1)	8.5 (3.6–13.1)	>0.05
Male [No. (%)]	5 (63%)	6 (50%)	>0.05
Original disease [No. (%)]
ALL	3 (37%)	5 (42%)	>0.05
AML	5 (63%)	7 (58%)	>0.05
CB cell dose
TNCs dose (×10⁷/kg), median (range)	6.80 (3.71–10.56)	7.21 (3.52–11.51)	>0.05
CD34⁺ cell dose (×10⁵/kg), median (range)	3.08 (2.59–6.11)	3.9 (2.8–10.9)	>0.05
Engraftment
Days to ANC > 0.5 × 10⁹/L, median (range)	12 (8–16)	21 (17–43)	0.003*
Days to platelet count > 20 × 10⁹/L, median (range)	30 (20–45)	73 (42–135)	0.004*
GVHD [No. (%)]
Acute GVHD, grades I–II	4 (50%)	7 (58%)	>0.05
Acute GVHD, grades III–IV	0 (0%)	1 (8%)	>0.05
Chronic GVHD, limited	1 (13%)	4 (33%)	>0.05
Chronic GVHD, extensive	0 (0%)	1 (8%)	>0.05
Outcome [No. (%)]
Transplant related mortality	2 (25%)	4 (33%)	>0.05
Overall survival rate	6 (75%)	8 (67%)	>0.05

ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ANC, absolute neutrophil count; CB, cord blood; CBT, cord blood transplantation; GVHD, graft-versus-host disease; TNCs, total nucleated cells; UCMSCs, umbilical cord-derived mesenchymal stem cells. *p < 0.05.

Characteristics of UCMSCs

Of the eight patients receiving cotransplantation of CB and UCMSCs, the range of HLA match between UCMSCs and recipients was 3/6 to 5/6. The median cell dose (×10⁶/ kg) and passage of UCMSCs were 7.19 (range, 2.44–10.12) and 3 (range, 2–4), respectively. The median duration of expansion to sufficient UCMSCs was 7 days (range, 5–8 days). Of the 8 units of UCMSCs, five UCMSC donors were female, and three were male. UCMSCs infused to the eight patients were shown to have a normal karyotype and the absence of pathogenic contamination after ex vivo expansion. The characteristics of UCMSCs are summarized in Table 1.

Safety of UCMSCs

The ex vivo expanded banked UCMSCs were infused during 30 min through a central venous line. The infusion of the UCMSCs was well tolerated by all recipients. There were no infusion-related toxicities such as allergic reactions, respiratory distress, or hypertensive responses. Of the eight patients receiving cotransplantation of CB and UCMSCs, no ectopic tissue was noted after MRI and PET survey in the interval of 3 months after UCMSC infusion. The median duration of follow-up after cotransplantation was 16.5 months (range, 8–27 months).

Engraftment, GVHD, and Outcome

All of the 20 patients, including the patients receiving cotransplantation and CBT only, achieved 100% donor chimerism assessed with the method of short tandem repeat on the 60th day after transplantation. Of the eight patients receiving cotransplantation of CB and UCMSCs, an ANC > 0.5 × 10⁹/L was seen after a median time of 12 days (range, 8–16 days) (Table 1). Platelets count > 20 × 10⁹/L was achieved at a median time of 30 days (range, 20–45 days) (Table 1) after cotransplantation. Of the 12 patients receiving CBT alone, an ANC > 0.5 × 10⁹/L was seen after a median time of 21 days (range, 17–43 days). Platelet count > 20 × 10⁹/L was achieved at a median time of 73 days (range, 42–135 days) after CBT. Patients receiving cotransplantion had faster recovery of ANC (p = 0.003) and platelets (p = 0.004) in comparison to those receiving CBT alone (Table 3).

None of the eight patients receiving cotransplantation encountered severe infection, including CMV disease, septic shock, or fungal infection. Of the eight patients receiving cotransplantation, four patients developed localized skin rashes (grades I–II acute GVHD), which responded to steroids. One of these four patients with acute GVHD developed limited chronic GVHD, which responded to long-term use of cyclosporine and steroids. Of the eight patients receiving cotransplantation, two patients expired due to leukemia relapse, and the other six patients remained alive and disease free with a median follow-up time of 16.5 months (range, 11–27 months). Of the 12 patients receiving CBT alone, one patient died from bacteremia due to Pseudomonas aeruginosa, one died from invasive pulmonary aspergillosis, and two patients died from leukemia relapse. The other eight patients remained alive and disease-free with a median follow-up time of 18.5 months (range, 12–31 months). The comparison between patients receiving cotransplantation and those receiving CBT alone is summarized in Table 3.

Discussion

In humans, BMMSCs are safe to infuse with no severe adverse events and no formation of ectopic tissue in clinical application (4,16,18–22,27,29). However, all the experiences about MSC safety are from BMMSCs. Amariglio et al. reported that a donor-derived brain tumor following neural tissue that included neural stem cells derived from fetal tissue was transplanted in an ataxia telangiectasis patient with immune deficiency (1). UCMSCs, one kind of fetal tissue-derived MSCs, are more primitive than BMMSCs. Although fetal tissue-derived MSCs are easier to obtain than BMMSCs and cause no suffering to the donor, the safety of these cells in human clinical application is still unknown but is likely to be very important. Few human clinical experiences of fetal tissue-derived MSCs have been reported until now (7,12,34). In the present study, after we infused banked UCMSCs from third-party HLA-mismatched donors to patients who were severely immunocompromised in the course of CBT, there was no infusion toxicity and no ectopic tissues detected by MRI and PET after 8–27 months follow-up. Therefore, we speculate that UCMCSs may be as safe as BMMSCs for clinical use, even in severe immunocompromised patients.

MSCs have low immunogenicity and do not express HLA class II (25). Le Blanc et al. found that the response rates of severe acute GVHD after BMMSC treatment were not related to the HLA match between recipients and BMMSCs (19). In the present study, after we infused 3/6 to 5/6 HLA-matched UCMSCs in the patients, no severe adverse effects were noted, and these cells enhanced hematopoiesis after CBT. Therefore, we speculate that the restriction of MSC matches between donors and recipients is less than other cells, such as HSCs. Besides, we found that UCMSCs proliferated faster than BMMSCs (34), as previously reported (3). In the present study, we could get an adequate cell dose of UCMSCs for recipients within 8 days, which is shorter than the time to get an adequate cell dose of BMMSCs for clinical use in the previous report (22). Therefore, UC can be considered as an alternative source of MSCs for clinical applications.

Gong et al. reported on banking UCMSCs for clinical use (12). MSCs for human clinical use should not only be strictly qualified for cell quality but must also be infection free. Therefore, all procedures of clinical-grade MSCs should be managed in a qualified manner, such as under GTP conditions. However, not all physicians have the facilities to guarantee the quality of cells for clinical applications. In this study, all UCMSCs provided from the UCMSC bank under GTP conditions were free of contamination. Therefore, MSC banks that cryopreserve manufactured unrelated MSCs may help solve time, donor variability, and contamination problems. Like CB banks (28), MSC banks may have a ready supply of “off-the-shelf” cells available for clinical use.

Chemotherapy and/or radiotherapy prior to HSCT damages the marrow stroma (10). MSCs play an important role in providing the specialized BM microenvironment and secret cytokines for hematopoiesis (33). Additionally, MSCs (8,15,25,31,35), including BMMSCs and UCMSCs, which we previously reported (34), possess immunosuppressive effects, so MSC infusion may suppress immune function in recipients and, therefore, enhance engraftment of donor HSCs. Cotransplantation of BMMSCs and HSCs was shown to promote engraftment after HSCT (4,16,18,21,22,27). In the present study, we found that patients receiving cotransplantation of CB and UCMSCs resulted in faster engraftment of ANC and platelets than those receiving CBT alone. Further studies are needed to confirm the cotransplantation of UCMSCs in the fast engraftment, the effect on GVHD, and long-term survival after CBT.

In conclusion, UCMSCs are easier to obtain without harm to the donor and proliferate faster than BMMSCs, which indicates that they may be ideal candidates for cell-based therapy. No severe adverse effects were noted in this human clinical application of UCMSCs. “Off-the-shelf” UCMSCs may make UCMSCs more available for clinical applications. Accordingly, application of ex vivo expanded banked UCMSCs in humans appears to be feasible and safe. Cotransplantation of UCMSCs was found to enhance hematopoiesis after CBT. Further studies are warranted.

Footnotes

Acknowledgments

This work was supported by the Ministry of Economic Affairs in Taiwan and grants from the China Medial University Hospital (DMR-102-035) and Chung Shan Medical University Hospital (CSH-2013-A-006). The authors declare no conflicts of interest.

References

Amariglio

Hirshberg

Scheithauer

B. W.

Cohen

Loewenthal

Trakhtenbrot

Paz

Koren-Michowitz

Waldman

Leider-Trejo

Toren

Constantini

Rechavi

Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient. PLoS Med. 6: 221–231; 2009.

Antonucci

Stuppia

Kaneko

Tajiri

Bae

E. C.

Chheda

S. H.

Weinbren

N. L.

Borlongan

C. V.

Amniotic fluid as a rich source of mesenchymal stromal cells for transplantation therapy. Cell Transplant. 20: 789–795; 2011.

Baksh

Yao

Tuan

R. S.

Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived umbilical cord and bone marrow. Stem Cells 25: 1384–1392; 2007.

Ball

L. M.

Bernardo

M. E.

Roelofs

Lankester

Cometa

Egeler

R. M.

Locatelli

Fibbe

W. E.

Cotransplantation of ex vivo expanded mesenchymal stem cells accelerates lymphocyte recovery and may reduce the risk of graft failure in haploidentical hematopoietic stem cell transplantation. Blood 110: 2764–2767; 2007.

Chang

J. W.

Hung

S. P.

H. H.

W. M.

Yang

A. H.

Tsai

H. L.

Yang

L. Y.

Lee

O. K.

Therapeutic effects of umbilical cord blood-derived mesenchymal stem cell transplantation in experimental lupus nephritis. Cell Transplant. 20: 245–257; 2011.

Chao

Y. H.

Peng

C. T.

Harn

H. J.

Chan

C. K.

K. H.

Poor potential of proliferation and differentiation in bone marrow mesenchymal stem cells derived from children with severe aplastic anemia. Ann. Hematol. 89: 715–723; 2010.

Chao

Y. H.

Tsai

Peng

C. T.

H. P.

Chan

C. K.

Weng

K. H.

Cotransplantation of umbilical cord MSCs to enhance engraftment of hematopoietic stem cells in patients with severe aplastic anemia. Bone Marrow Transplant. 46: 1391–1392; 2011.

Deuse

Stubbendorff

Tang-Quan

Phillips

Kay

M. A.

Eiermann

Phan

T. T.

Volk

H. D.

Reichenspurner

Robbins

R. C.

Schrepfer

Immunogenicity and immunomodulatory properties of umbilical cord lining mesenchymal stem cells. Cell Transplant. 20: 655–667; 2011.

Ding

D. C.

Shyu

W. C.

Lin

S. Z.

Mesenchymal stem cells. Cell Transplant. 20: 5–14; 2011.

10.

Galotto

Berisso

Delfino

Podesta

Ottaggio

Dallorso

Dufour

Ferrara

G. B.

Abbondandolo

Dini

Bacigalupo

Cancedda

Quarto

Stromal damage as consequence of high-dose chemo/radiotherapy in bone marrow transplant recipients. Exp. Hematol. 27: 1460–1466; 1999.

11.

Gluckman

History of cord blood transplantation. Bone Marrow Transplant. 44: 621–626; 2009.

12.

Gong

Han

Zhao

Wang

Zhong

Wang

Sun

Han

Banking human umbilical cord derived mesenchymal stromal cells for clinical use. Cell Transplant. 21: 207–216; 2012.

13.

Hunt

D. P.

Irvine

K. A.

Webber

D. J.

Compston

D. A.

Blakemore

W. F.

Chandran

Effects of direct transplantation of multipotent mesenchymal stromal/stem cells into the demyelinated spinal cord. Cell Transplant. 17: 865–873; 2008.

14.

Iop

Chiavegato

Callegari

Bollini

Piccoli

Pozzobon

Rossi

C. A.

Calamelli

Chiavegato

Gerosa

De Coppi

Sartore

Different cardiovascular potential of adult- and fetal-type mesenchymal stem cells in a rat model of heart cryoinjury. Cell Transplant. 17: 679–694; 2008.

15.

Keyser

K. A.

Beagles

K. E.

Kiem

H. P.

Comparison of mesenchymal stem cells from different tissues to suppress T-cell activation. Cell Transplant. 16: 555–562; 2007.

16.

Koç

O. N.

Gerson

S. L.

Cooper

B. W.

Dyhouse

S. M.

Haynesworth

S. E.

Caplan

A. I.

Lazarus

H. M.

Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J. Clin. Oncol. 18: 307–316; 2000.

17.

Koldehoff

Steckel

N. K.

Hlinka

Beelen

D. W.

Elmaagacli

A. H.

Quantitative analysis of chimerism after allogeneic stem cell transplantation by real-time polymerase chain reaction with single nucleotide polymorphisms, standard tandem repeats, and Y-chromosome-specific sequences. Am. J. Hematol. 81: 735–746; 2006.

18.

Lazarus

H. M.

Koc

O. N.

Devine

S. M.

Curtin

Maziarz

R. T.

Holland

H. K.

Shpall

E. J.

McCarthy

Atkinson

Cooper

B. W.

Gerson

S. L.

Laughlin

M. J.

Loberiza

F. R.

Moseley

A. B.

Jr. Bacigalupo

Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients. Biol. Blood Marrow Transplant. 11: 389–398; 2005.

19.

Le Blanc

Frassoni

Ball

Locatelli

Roelofs

Lewis

Lanino

Sundberg

Bernardo

M. E.

Remberger

Dini

Egeler

R. M.

Bacigalupo

Fibbe

Ringdén

Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: A phase II study. Lancet 371: 1579–1586; 2008.

20.

Le Blanc

Rasmusson

Sundberg

Götherström

Hassan

Uzunel

Ringdén

Treatment of severe acute graft-versus-host disease with third-party haploidentical mesenchymal stem cells. Lancet 363: 1439–1441; 2004.

21.

Le Blanc

Samuelsson

Gustafsson

Remberger

Sundberg

Arvidson

Ljungman

Lönnies

Nava

Ringdén

Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells. Leukemia 21: 1733–1738; 2007.

22.

Macmillan

M. L.

Blazar

B. R.

DeFor

T. E.

Wagner

J. E.

Transplantation of ex-vivo culture-expanded parental haploidentical mesenchymal stem cells to promote engraftment in pediatric recipients of unrelated donor umbilical cord blood: Results of a phase I–II clinical trial. Bone Marrow Transplant. 43: 447–454; 2009.

23.

Magatti

De Munari

Vertua

Nassauto

Albertini

Wengler

G. S.

Parolini

Amniotic mesenchymal tissue cells inhibit dendritic cell differentiation of peripheral blood and amnion resident monocytes. Cell Transplant. 18: 899–914; 2009.

24.

Marchetti

Santangelo

Manzara

D'Onghia

Fadda

Cattani

Comparison of real-time PCR and pp 65 antigen assays for monitoring the development of Cytomegalovirus disease in recipients of solid organ and bone marrow transplants. New Microbiol. 34: 157–164; 2011.

25.

Nauta

A. J.

Fibbe

W. E.

Immunomodulatory properties of mesenchymal stromal cells. Blood 110: 3499–3506; 2007.

26.

Pittenger

M. F.

Mackay

A. M.

Beck

S. C.

Jaiswal

R. K.

Douglas

Mosca

J. D.

Moorman

M. A.

Simonetti

D. W.

Craig

Marshak

D. R.

Multilineage potential of adult human mesenchymal stem cells. Science 284: 143–147; 1999.

27.

Poloni

Leoni

Buscemi

Balducci

Pasquini

Masia

M. C.

Viola

Costantino

Discepoli

Corradini

Tagliabracci

Olivieri

Engraftment capacity of mesenchymal cells following hematopoietic stem cell transplantation in patients receiving reduced-intensity conditioning regimen. Leukemia 20: 329–335; 2006.

28.

Querol

Rubinstein

Marsh

S. G.

Goldman

Madrigal

J. A.

Cord blood banking: “Providing cord blood banking for a nation.” Br. J. Haematol. 147: 227–235; 2009.

29.

Ringdén

Uzunel

Rasmusson

Remberger

Sundberg

Lönnies

Marschall

H. U.

Dlugosz

Szakos

Hassan

Omazic

Aschan

Barkholt

Le Blanc

Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease. Transplantation 81: 1390–1397; 2006.

30.

Rubinstein

Carrier

Scaradavou

Kurtzberg

Adamson

Migliaccio

A. R.

Berkowitz

R. L.

Cabbad

Dobrila

N. L.

Taylor

P. E.

Rosenfield

R. E.

Stevens

C. E.

Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N. Engl. J. Med. 339: 1565–1577; 1998.

31.

Sattler

Steinsdoerfer

Offers

Fischer

Schierl

Heseler

Däubener

Seissler

Inhibition of T cell proliferation by murine multipotent mesenchymal stromal cells is mediated by CD39 expression and adenosine generation. Cell Transplant. 20: 1221–1230; 2011.

32.

Secco

Zucconi

Vieira

N. M.

Fogaça

L. L.

Cerqueira

Carvalho

M. D.

Jazedje

Okamoto

O. K.

Muotri

A. R.

Zatz

Multipotent stem cells from umbilical cord: Cord is richer than blood!

Stem Cells 26: 146–150; 2008.

33.

Verfaillie

C. M.

Direct contact between human primitive hematopoietic progenitors and bone marrow stroma is not required for long-term in vitro hematopoiesis. Blood 79: 2821–2826; 1992.

34.

K. H.

Chan

C. K.

Tsai

Chang

Y. H.

Sieber

Chiu

T. H.

Peng

C. T.

H. P.

Huang

J. L.

Effective treatment of severe steroid-resistant acute graftversus-host disease with umbilical cord-derived mesenchymal stem cells. Transplantation 91: 1412–1416; 2011.

35.

Yagi

Soto-Gutierrez

Parekkadan

Kitagawa

Tompkins

R. G.

Kobayashi

Yarmush

M. L.

Mesenchymal stem cells: Mechanisms of immunomodulation and homing. Cell Transplant. 19: 667–679; 2010.