Clin Exp Med. 2015 Feb;15(1):41-5.

Novel therapy for insulin-dependent diabetes mellitus: infusion of in vitro-generated insulin-secreting cells.

S D Dave1, A V Vanikar2, H L Trivedi 1, 3, U G Thakkar 3, S Chooramani4, T Chandra5

  1. Department of Stem Cell Therapy and Regenerative Medicine, IKDRC-ITS, Ahmedabad, Gujarat, India
  2. Department of Pathology, Laboratory Medicine, Transfusion Services and Immunohematology, IKDRC-ITS, Ahmedabad, Gujarat, India
  3. Department of Nephrology and Transplantation Medicine, IKDRC-ITS, Ahmedabad, Gujarat, India
  4. Department of surgery, Banaras Hindu University, Varanasi, Uttar Pradesh, India
  5. Department of Transfusion Medicine, King George Medical University, Lucknow, Uttar Pradesh, India

Correspondence Address

Shruti D. Dave, Ph.D.

Senior Scientific Officer, Department of Stem Cell Therapy and Regenerative Medicine,

G.R. Doshi and K. M. Mehta Institute Of Kidney Diseases & Research Centre (IKDRC)- Dr. H.L. Trivedi Institute Of Transplantation Sciences (ITS),

Civil Hospital Campus, Asarwa, Ahmedabad- 380016,

Tele : 0091 79 2268 5600/7439

Fax : 0091 79 2268 5454

Email – shrutiddave@yahoo.com

 

Abstract

Stem cell therapy is promising avenue for management of many autoimmune disorders/ diseases including insulin dependent diabetes mellitus (IDDM). Mesenchymal stem cells (MSC) possess immunomodulatory properties. They can be isolated from various sources including adipose tissue and can be differentiated in to insulin-secreting cells (ISC).

We present our experience of treating IDDM patients with co-infusion of in vitro generated ISC differentiated from MSC generated from patient’s own (autologous) adipose tissue along with bone marrow (BM) derived hematopoietic stem cells (HSC). This was an Institutional Review Board approved prospective non-randomized open-labeled clinical trial after informed consent from 10 patients. ISC and HSC were infused in subcutaneous tissue, portal and thymic circulation. Patients were monitored for blood sugar levels, serum C-peptide levels, glycosylated hemoglobin (HbA1c) and glutamic acid decarboxylase (GAD) antibodies. Insulin was administered as per sliding scale with an objective of maintaining fasting blood sugar (FBS) <150 mg/dL and postprandial blood sugar (PPBS) around 200 mg/dL.

Over a mean follow-up of 31.71 months, mean serum C-peptide of 0.22 ng/ml before infusion had sustained rise of 0.92 ng/ml with decreased exogenous insulin requirement from 63.9 international units (IU)/day to 38.6 IU/day. Mean HbA1c was dropped from 10.99 % to 6.72 %. Mean GAD antibodies were positive in all patients with mean of 331.10 IU/ml, which decreased to mean of 123 IU/ml.

Thus co-infusion of autologous ISC with HSC represents a viable novel therapeutic option for management of IDDM.

PMID: 24317657

 

Supplement

IDDM also known as type 1 DM (T1DM) or juvenile diabetes is a form of DM that results from autoimmune destruction of insulin-producing beta-cells of the pancreas. It is characterized by loss of insulin-producing beta-cells of the islets of Langerhans of pancreas, leading to insulin deficiency. IDDM is believed to be immune-mediated, where beta-cell loss is a T-cell mediated autoimmune attack with a potential risk of developing ketoacidosis, coma and eventually death if not attended immediately. IDDM is usually characterized by the presence of anti-GAD (glutamic acid decarboxylase), anti- islet cell or anti-insulin antibodies which identify the autoimmune processes that lead to beta-cell destruction. Potential therapy for IDDM needs to address insulin-replacement and immune dys-regulation arising in these patients.

This was a prospective non-randomized open-labeled clinical trial conducted from January 2010 to January 2013 to test efficacy and safety of infusion of in vitro generated autologous adipose tissue derived MSC differentiated ISC, combined with in vitro generated BM derived HSC to treat IDDM patients. It was approved by the Institutional Review Board after informed consent in 10 patients (9 males, 1 female) as insulin replacement therapy. Inclusion criteria were patients between 8 to 45 years with confirmed diagnosis of IDDM at least of 6 months duration, with low levels of serum C-peptide levels (<0.5 ng/mL). Patients themselves were approved as donors.

MSC were generated as per our previously described technique from adipose tissue, harvested on day 10, further differentiated into ISC on day 14, quantified and tested for sterility, viability and insulin-secreting markers (Pax-6, Ipf-1 and Isl-1) by immunofluorescence. C-peptide and insulin secretion were tested by chemiluminescence assay (Lumax, USA). Glucose sensitivity assay was also carried out [1].

Ten IDDM patients (9 males, 1 female) with mean age 20.2 years (range: 9–29 years) with mean disease duration of 8.1 years (range: 2–15 years) were subjected for infusion. Key endpoints of study were morbidity, mortality, untoward side effects from stem cell infusion, and changes in exogenous insulin requirements. Patients were conditioned with bortezomib, 1.3 mg/m2 body surface area along with methylprednsione, and 125 mg intravenously in normal saline (250 ml) on days 1, 4, 8 and 11 followed by rabbit anti-thymocyte globulin, 1.5 mg/kg BW on day 13. Key endpoints of study were morbidity, mortality, untoward side effects from stem cell infusion, and changes in exogenous insulin requirements. Secondary endpoints were serum C-peptide, GAD antibodies and HbA1c following infusion. Cells were infused in subcutaneous tissue, portal and thymic circulation. Thymic infusion was carried out to induce central tolerance [2] and peripheral tolerance was achieved by portal infusion, liver being the most tolerogenic organ of the body [3]. The method was indigenously designed as described previously [4].

Analysis of Infused Cells

Mean quantum of ISC was 3.34 ml with mean cell count of 5.25 x104/µl. Mean HSC quantum was 103.5 ml; with mean cell count of 2.66 x 106/µl. Mean HSC count was 60.55 X 106/Kg BW (range: 17.4 X 106– 149 X 106) and ISC count was 2.7 X 104/Kg BW (range: 0.38 X 104– 6.6 X 104). ISC expressed presence of transcription factors, Pax-6, Ipf-1 and Isl-1. Mean C-peptide and insulin secretion level of in vitro generated insulin-secreting cells was 1.03 ng/mL and 17.48 μ IU/L respectively after two hours of incubation in glucose medium following glucose sensitivity assay.

Patient status

There was no untoward effect of stem cell infusion. Over a mean follow-up of 31.71 months, improvement was sustained in all patients till the last follow-up. Interestingly all of them have rehabilitated to normal life style and normal unrestricted diet.

Pre-infusion mean serum C-peptide levels of 0.22 ng/mL increased gradually to mean 0.92 ng/mL (reference range: 0.7-1.9 ng/mL) (Monobind Inc, USA). Improvement in HbA1c was observed in the form reduction of mean 10.99 % to 6.72 % post-infusion. Gradual fall in exogenous insulin requirement was observed from pre-infusion requirement of 63.9 IU/day to 38.6 IU /day. Stable decline in FBS was observed with mean 197.2 mg/dL from mean 269.6 mg/dL and mean and mean PPBS drop of 261 mg/dL was noted from mean 372 mg/dL which remained sustained till last follow-up. Anti-GAD antibodies were positive in all patients with mean of 331.10 IU/ml before the initiation of therapy, which decreased to mean of 123 IU/ml. Six out of 10 patients had associated 6.5 DKA episodes before the treatment; which disappeared in all of them along with subjective well-being and mean weight gain to reach 53.05 kg from baseline of mean 51.47 kg before the treatment. None of the patient was administered any immunosuppressive medication at any time of therapy or following therapy since the source of stem cells was autologous.

We believe that the results demonstrated in this study provide evidence supporting the notion that differentiation of autologous human adipose tissue derived MSC to insulin-producing cells represent a viable therapeutic option for IDDM without use of any immunosuppression. The major advantage of this study is that it throws light on availability of sources other than embryonic derived / allogeneic stem cells and alternative medications to control IDDM. To our knowledge this is the first report of in vitro generation of insulin making cells from mesenchymal stem cells derived from adipose tissue and also effectively and safely using them in clinic. Thus it opens up avenues for IDDM.

 

Abbreviations

BM: bone marrow
DKA: diabetic ketoacidosis
FBS: fasting blood sugar levels
GAD: glutamic acid decarboxylase
Hb1Ac: glycosylated hemoglobin
HSC: hematopoietic stem cells
ISC: insulin-secreting cells
MSC: mesenchymal stem cells
PPBS: postprandial blood sugar level
IDDM: insulin dependent diabetes mellitus

 

Conflict Of Interest: None

Financial Support: None

Acknowledgments

The authors thank Mr. C N Patel and Mr. J.V Patel for technical help in stem cell lab and flowcytometry.

 

References

  1. Dave SD, Vanikar AV and Trivedi HL. Ex vivo generation of glucose sensitive insulin secreting mesenchymal stem cells derived from human adipose tissue. Indian J Endocr Metab 2012; 16(1): S65-S69.
  2. Starzl TE. The “Privileged” Liver and Hepatic Tolerogenicity. Liver Transpl 2001; 7(10): 918–920.
  3. SprentJ and H Kishimoto. The thymus and central tolerance. Philos Trans R Soc Lond B Biol Sci 2001; 29 356(1409): 609–616.
  4. Vanikar AV, Dave SD, Thakkar UG, Trivedi HL (2010) Co-transplantation of adipose tissue-derived insulin-secreting mesenchymal stem cells and hematopoietic stem cells: A novel therapy for insulin-dependent diabetes mellitus. Stem Cells International; Article ID 582382 5 pages.

 

 

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