Amniotic stem cell transplantation therapy for type 1 diabetes: A case report

Abstract

This case report presents an evaluation of the clinical effects of an allogeneic amniotic cell transplant for the treatment of type 1 diabetes mellitus. A 26-year-old man with type 1 diabetes was treated with stem cells isolated from his neonatal son’s amniotic membrane, collected at birth (2 × 10⁷ cells). The cells, which expressed high levels of cluster of differentiation (CD) 133 and CD34 as assessed by flow cytometry, were infused into the pancreatic dorsal artery through the left femoral artery. The main study outcome was the change in exogenous insulin requirements, which began to decrease 3 days after transplantation. At 3 months post-transplantation, the patient was insulin independent and remained so for 6.2 months. During a 36-month follow-up, the patient’s blood glucose remained under control and insulin treatment was readjusted to a dosage of 8 IU/day. These preliminary data suggest that amniotic membrane stem cell transplantation can improve islet-cell function in response to glucose in vivo, although an alternative explanation (such as a honeymoon period due to reduced glucose toxicity) also has to be considered.

Keywords

Type 1 diabetes mellitus amniotic stem cell transplantation insulin

Introduction

Diabetes mellitus is becoming one of the main threats to human health.^1,2 The global prevalence of diabetes is shifting substantially from the developed world to developing countries.^3–5 β-cell replacement is an effective treatment for type 1 diabetes; however, it is limited by the availability of sufficient donor tissue, as well as by the immunosuppressive effects of therapeutic regimens, which are used as adjuvant treatments.^6–8 Replacing lost β cells with new functional insulin-producing β cells in the pancreatic islets of Langerhans represents a novel alternative therapeutic strategy that could permit improvements in health-related quality of life and potentially provide a cure for patients with type 1 diabetes. The purposes of this case report are to raise awareness of the potential of amniotic stem cell transplantation for the treatment of type 1 diabetes and to report our clinical experience in one case.

Case report

A 26-year-old man, diagnosed 1 month prior with type 1 diabetes, was admitted to the Stem Cell Clinical Application Centre, Siping Central Hospital, Siping, Jilin Province, China, for stem cell transplantation in September 2009. As his wife was due to deliver a baby 1 month later, the decision was made to perform stem cell transplantation using his newborn child’s amniotic membrane stem cells, which were prepared as described below. Before transplantation, the patient’s blood glucose level was controlled by regular Novolin® injections (38 IU/day; Novo Nordisk, Beijing, China). His fasting blood glucose (FBG) was between 6.5–7.5 mmol/l; his 2-h postprandial blood glucose (2-h PG) was controlled between 8.0–10.0 mmol/l and was stable for 5 days in preparation for the transplantation. The quantity of regularly administered insulin at this time was considered to be the basal dosage for the patient.

When the patient’s son was born, five human amniotic membrane samples of similar size were collected after written informed consent was obtained from the mother, in accordance with the Ethical Committee of Siping Central Hospital. From each sample, 8 × 8 cm² sections of amniotic membrane were obtained. They were washed with 1.0 M phosphate-buffered saline (PBS; pH 7.2) containing 300 IU/ml penicillin and 300 µg/ml streptomycin (Gibco, Grand Island, NY, USA), and were immediately immersed in Dulbecco’s modified Eagle’s medium (DMEM)-high glucose (Gibco), supplemented with 10% fetal bovine serum (FBS; Gibco), 300 IIU/ml penicillin and 300 µg/ml streptomycin. All samples were processed within 12–15 h after collection.

The amniotic membranes were treated with 0.1% collagenase I (Sigma-Aldrich, St Louis, MO, USA) in 1.0 M PBS (pH 7.2) and were incubated at 37℃ for 20 min. Each amniotic membrane was washed three times with low-glucose DMEM (Gibco), and the detached cells were harvested after a gentle massage of the amniotic membrane. The cells were centrifuged at 300 g for 10 min at 37℃, resuspended in RPMI 1640 medium with 10% FBS, then grown in 25 cm² flasks at a density of 1 × 10⁶ cells/ml. After 24 h incubation, nonadherent cells were removed. The culture medium was replaced every 3 days. Adherent cells were cultured until they reached 80–90% confluence.

To analyse the distribution of typical protein markers (for example, the haematopoietic lineage markers cluster of differentiation [CD] 45 and human leucocyte antigen [HLA]-DR, and the cell-surface markers of endothelial progenitor cells CD133 and CD34 on the surfaces of amniotic membrane-derived cells), adherent cells were incubated for 2 h at 37℃ with the rabbit antihuman fluorophore-conjugated primary antibodies (all diluted 1 : 1000 in 0.01 M PBS; all from Becton Dickinson, Franklin Lakes, NJ, USA) for direct immunofluorescence staining. A total of 10 000 labelled cells were analysed using a Becton Dickinson LSRFortessa™ Cell Analyzer, running Becton Dickinson FACSDiva™ software version 6.0 (Becton Dickinson Biosciences, San Jose, CA, USA).

Primary adherent cultures were generated from all amniotic membrane samples, with cells displaying a mesenchymal stem cell (MSC)-like phenotype. After 4 days in culture the cells formed colonies, reaching confluence after 10–14 days. Most of the cells were spindle-shaped, resembling fibroblasts. After the second passage, adherent cells constituted homogeneous cell layers with an MSC-like phenotype (Figure 1). The number of adherent cells from the amniotic membrane decreased slightly after freezing and thawing, and the remaining viable cells were successfully expanded on consecutive days (data not shown).

Figure 1.

Representative light photomicrographs of cultured adherent cells, isolated from the amniotic membrane of a male neonate, in preparation for transplantation into his father for correction of newly diagnosed type 1 diabetes mellitus. (A) Samples of amniotic membrane generated primary adherent cultures, with cells displaying a mesenchymal stem cell-like phenotype. After 4 days in culture, these cells formed colonies (B). Cells reached confluence after 10–14 days with most cells being spindle shaped. Original magnification ×40.

Very few of the adherent cells derived from the amniotic membrane had haematopoietic lineage markers CD45 and HLA-DR (HLA-class II) (Figure 2) on the cell surface, as assessed by flow cytometry. The majority of the cells presented high levels of CD133 and CD34 proteins on the cell surface (Figure 2). In comparison with the fibroblast control, no obvious difference in the levels of surface antigens could be observed (data not shown).

Figure 2.

Flow cytometric analyses of cell surface protein presentation on adherent cells, from the amniotic membrane from a male neonate, in preparation for transplantation into his father for correction of newly diagnosed type 1 diabetes mellitus. The majority of adherent cells derived from the amniotic membrane did not have cluster of differentiation (CD) 45 proteins on their cell surface (A), but the majority of cells had high levels of CD34 protein (B). None of the adherent cells derived from the amniotic membrane had human leucocyte antigen (HLA)-DR proteins (C), but the majority of cells had high levels of CD133 (D). Solid line, negative control sample; dashed line, adherent cells from the amniotic membrane.

Fifteen days after the purification and amplification of his son’s amniotic stem cells, these cells (passage 5, 2 × 10⁷ cells) were suspended in saline (10 ml) and used for transplantation, as follows. The patient was placed in the supine decubitus position on the operating table and his left groin was disinfected with iodophor. Under direct observation, a catheter was inserted into the left external iliac artery, after local anaesthesia (10% lidocaine) was administered subcutaneously. Amniotic stem cells suspended in saline (2 × 10⁷ cells in 10 ml) were injected into the pancreatic dorsal artery through the left femoral artery. The puncture catheter was withdrawn and a sterilized pressure bandage was wrapped around the puncture site. The patient lay supine on the operating table for a further 30 min and was then sent to a single-person ward. The antibiotic regimen of penicillin V sodium (Hangzhou Xinxing Pharmaceutical Factory, Hangzhou, China), 0.25 g, was administered orally three times daily for 7 days, to help prevent infection. The following parameters were monitored in the patient at 3, 6, 12, 24 and 36 months after transplantation, using standard laboratory methods: fasting insulin (FIN); FBG; glycosylated haemoglobin (HbA_1c); 2-h PG, plasma triglyceride (TG); total cholesterol (TC); low-density lipoprotein cholesterol (LDL-C); high-density lipoprotein cholesterol (HDL-C). The patient follow-up period continued for 36 months post-transplantation.

As shown in Table 1, the patient’s FIN increased significantly post-transplantation (P < 0.05), while FBG and 2-h PG levels decreased significantly compared with the pretransplant values (P < 0.05 for both). Body mass index, waist circumference and hip circumference increased, although the differences were not significant. The waist-to-hip ratio remained similar over the 36-month follow-up period. There were no significant changes in HbA_1c, TG, TC, HDL-C and LDL-C after transplantation.

Table 1.

Clinical data for a 26-year-old male patient with newly diagnosed type 1 diabetes mellitus, before and after he received a transplantation of adherent stem cells derived from the amniotic membrane collected from his neonatal son.

Characteristic	Pretransplantation	Post-transplantation, months
Characteristic	Pretransplantation	3	6	12	24	36
FBG, mmol/l^a	7.61 ± 0.41	7.13 ± 0.63*	5.93 ± 0.27*	6.22 ± 0.31*	5.82 ± 0.42*	6.03 ± 0.19*
FIN, IU^a	2.14 ± 0.14	6.22 ± 0.33*	6.42 ± 0.21*	5.84 ± 0.12*	5.21 ± 0.17*	5.61 ± 0.15*
BMI, kg/m^2a	23.53 ± 3.22	24.51 ± 2.14	24.34 ± 1.89	25.52 ± 2.07	24.83 ± 1.66	24.12 ± 2.37
WC, cm	87	93	96	98	96	95
HC, cm	113	124	127	131	127	125
Waist-to-hip ratio	0.77	0.75	0.76	0.75	0.76	0.76
HbA_1c, %	7.72	6.63	6.12	6.38	7.03	6.29
2-h PG, mmol/l^a	10.42 ± 1.13	10.15 ± 0.83	8.33 ± 0.36*	7.95 ± 0.47*	8.09 ± 0.56*	8.42 ± 0.61*
TG, mmol/l	2.05	2.12	2.14	2.23	2.18	2.09
TC, mmol/l	4.97	4.89	4.76	4.63	4.82	4.78
HDL-C, mmol/l	2.31	2.51	2.46	2.59	2.43	2.52
LDL-C, mmol/l	2.72	2.64	2.61	2.59	2.64	2.62

Data expressed as mean ± SD of five samples; all other values measured once at each timepoint.

P < 0.05 compared with pretransplant value; one-way analysis of variance and Student’s t-test.

FBG, fasting blood glucose; FIN, fasting insulin; BMI, body mass index; WC, waist circumference; HC, hip circumference; HbA_1c, glycosylated haemoglobin; 2-h PG, 2-h postprandial blood glucose; TG, triglyceride; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.

As shown in Table 2, there was a decrease in the quantity of insulin required beginning at day 3 after transplantation, with a complete withdrawal of insulin occurring after 3 months. The patient’s blood glucose level increased again 6.2 months after complete withdrawal of insulin. After readjusting his insulin treatment, the required dose of insulin was 8 IU/day, with blood glucose levels remaining under control for the remaining duration of the follow-up period. No adverse events were observed in the patient during the follow-up period.

Table 2.

Insulin usage in a 26-year-old male patient with type 1 diabetes mellitus, before and after he received a transplantation of adherent cells derived from the amniotic membrane collected from his neonatal son.

Parameter	Value
Pretransplant insulin dosage, IU/day	38
Time following surgery to the start of reduced insulin therapy, days	3
Time when insulin therapy discontinued, months	3
Total time free of insulin therapy, months	6.2
Dosage of insulin after readjustment at 12 months, IU/day	8
Dosage of insulin after readjustment at 24 months, IU/day	8
Dosage of insulin after readjustment at 36 months, IU/day	8

Discussion

The present case report investigated the potential use of cell culture-expanded undifferentiated amniotic stem cells for a cell-based treatment strategy in a patient with type 1 diabetes. It is difficult to obtain an adequate number of human stem cells: there are a variety of reasons for this, including technical difficulties associated with the isolation of normally occurring populations of stem cells in adult tissues, the high cost and very low yields achieved, and the ethical and moral issues of obtaining such cells from embryos or fetal tissue. The present case report described the use of amniotic membrane as a potential source of stem cells, which is a novel strategy for obtaining stem cells to treat type 1 diabetes.

The placenta, which contains cells with immunomodulatory properties, maintains fetal tolerance.^9–11 The use of placental tissue does not elicit ethical debate, as it is readily available and easily procured. Although the immunomodulatory properties are not yet completely understood, the amniotic membrane is a promising new source of stem cells. In the present case report, amniotic membrane cells were successfully isolated from the human chorionic villi of a full-term placenta. The chorionic villi of the human full-term placenta are a rich source of MSCs.¹² These amniotic membrane cells have a typical fibroblast-like appearance; different human amniotic cells have the ability to treat different diseases using different mechanisms of action.^13–19 Cells that were used in this present case report were different from MSCs, as the majority of the cells had high levels of CD133 and CD34 on their cell surfaces (as shown by flow cytometry). CD133 and CD34 are considered to be the cell surface markers of endothelial progenitor cells.²⁰ Therefore, the improved pancreatic islet function observed in this current case post-transplantation may be attributed to new blood vessel formation. Although the patient subsequently required insulin to control his blood glucose levels, he was not dependent on insulin injections for 6.2 months. Even when the patient required insulin therapy to be reintroduced, his daily insulin requirement had decreased considerably (from 38 IU/day to 8 IU/day; Table 2), suggesting an improvement in β-cell function. In addition, the patient experienced no adverse effects of transplantation, during the 36-month follow-up period.

Several studies have reported on the treatment of newly diagnosed type 1 diabetes using stem cell transplantation. For example, newly diagnosed type 1 diabetes patients treated with autologous haematopoietic stem cell transplantations achieved good glycaemic control.^21,22 Polgreen et al.²³ reported that the majority of children with Fanconi anaemia had normal glucose tolerance and normal β-cell function after haematopoietic cell transplantation. Another study, which used human adipose tissue-derived insulin-making MSC transfused with unfractionated cultured bone marrow to treat five patients with insulinopenic diabetes, showed a 30–50% decrease in insulin requirements with a 4- to 26-fold increase in serum C-peptide levels.²⁴ This current case report describes a well-tolerated and effective treatment for type 1 diabetes, using amniotic stem cells.

One limitation of this current case report is the lack of C-peptide data, which could have confirmed a direct positive effect of stem cell treatment on β-cell function rather than a prolonged honeymoon effect resulting from close medical follow-up and physician-directed changes in lifestyle. Further randomized controlled clinical trials, involving more patients, will be required to confirm our preliminary findings and to elucidate the biological processes involved in improving glycaemic control, following transplantation with undifferentiated amniotic stem cells.

In conclusion, this current case report demonstrated that amniotic membrane stem cells can be easily isolated and expanded in vitro, without alterations to their morphological and functional characteristics. Due to their easy procurement, lack of ethical concerns and abundant availability, amniotic membrane stem cells may be an attractive alternative source of progenitor/stem cells for basic or translational research. These current data show that amniotic membrane stem cell transplantation may improve islet function in response to glucose in vivo, but it should be noted that full recovery of β-cell function was not achieved in the long term in this patient; exogenous insulin treatment was required, several months after transplantation.

Footnotes

Acknowledgements

We want to thank Dr Leonid Poretsky, Division of Endocrinology and Metabolism, Beth Israel Medical Center, Albert Einstein College of Medicine, New York, NY, USA, who reviewed this manuscript.

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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