Stem cell therapy for type1 diabetes with transplantation of stem cells into the Omental pouch, peritoneum, and blood, experimental study.

Introduction: Type 1 diabetes is a dreadful autoimmune disease of childhood with incidence of 0.26/1000 children in India. To develop a new, cheap, and effective treatment for this disease, we invented an autologous Stem cell therapy for type 1 diabetes in which stem cells are transplanted into the Omental pouch, peritoneum, and blood. The Omental pouch stem cell operation in the therapy is reported for the first time in the medical literature. Materials and methods: Last 5 years I treated 21 patients of Type 1 diabetes with autologous stem cell therapy and in the same period, a group of 26 patients of Type 1 diabetes with conventional treatment of Insulin injections was put as a control group. Blood sugar fasting and post prandial, Anti Gad antibody titer, Glycosylated Hb and C peptide levels and weight of patient and total Insulin requirement in 24 h were the variables to be measured before the therapy and after the therapy. Stem cells were harvested from patients own bone marrow and separated by density gradient method. An infusion of 20 mg/kg methylprednisolone in 100 ML normal saline given intravenously over 1 h prior to the therapy. The total average numbers of cells harvested were 7.86 × 107. One third quantity of isolated stem cells were put into the Omental pouch through no. 7 IFT, another one third into peritoneal cavity through no. 10 IFT and remaining third is given IV in 100 ml normal saline.
Results: The minimum follow up was 6 months and maximum of 4 years. In the therapy group, the average weight gain after one year of therapy, daily requirement of Insulin and its drop after therapy, drop in HbA1c levels, drop in fasting and post prandial blood sugar, rise of C peptide levels and drop in Anti-GAD antibody titer were measured and was found to be statistically highly significant. The same parameters were measured in control group and was not statistically significant. There were a few side effects noted after stem cell therapy such as mild skin rash, nausea, and pain in abdomen. Discussion: In autologous bone marrow derived stem cell therapy, cells are transplanted into the Omental pouch, peritoneum, and blood. Cells transplanted in the Omental pouch get vascularized like a split skin graft. Omental surface has far less cellular immunity than blood, hence, if some of these cells get converted into Islets like cells producing Insulin, then they are less vulnerable to damage by the immune system. It means that the Omental pouch may act as a new biological pancreas producing Insulin. Stem cells injected intravenously reach the pancreases and may get differentiate into Islet like cells due to specific growth factors released by pancreas. Stem cells can reverse autoimmunity by their immunomodulatory function. Stem cells transplanted in peritoneum grow longer due to large surface area and little cellular immunity and secrete growth factors and cytokines for a long time which can rejuvenate existing Islets of Langerhans. The therapy group had substantially good results compared to the control group and the difference was statistically highly significant. Conclusions: Autologous stem cell therapy was safe, and effective for the long term for the treatment of Type 1 diabetes. We need a greater number of cases and a longer follow up to make it better. The therapy creates a lot of hope for Type 1 diabetes patients as it can be easily repeated any number of times.

Introduction

Type 1 diabetes is a dreadful autoimmune disease of childhood with incidence of 0.26/1000 children in India [1]. Currently insulin injection and islet cell transplantation are the FDA approved treatments. Taking multiple insulin shots daily and its cost are both unacceptable to most of the children. Islet cell transplantation is exorbitantly costly and gives relief only for 2–5 years [2]. Medical literature has many promising animals and in Vitro studies published where stem cells have established euglycemia in diabetic rats. We need to bring the findings of animal studies on human side for the welfare of Type 1 diabetes patients. A new, cheap, and effective method needs to be developed for the treatment of this disease. There are different benefits of implanting stem cells in various parts of the body by different routes. Hence, we invented autologous bone marrow derived stem cell therapy for type 1 diabetes with transplanted of stem cells into the Omental pouch, peritoneum, and blood which gives the benefits of all 3 methods to the patient. The Omental pouch operation is reported for the first time in the medical literature (see Fig. 1).

Figure:1

Showing the Omental pouch with no. 7 infant feeding tube.

Materials and methods: study design

Last 5 years I treated 21 patients of Type 1 diabetes with stem cell therapy in which autologous bone marrow derived stem cells were implanted into an Omental pouch, peritoneum, and blood. Age range in the therapy group was 3.5 years to 38 years. In the same period, a group of 26 patients of type 1 diabetes with conventional treatment of Insulin injections was put as a control group. Patients were distributed in both the groups in a random way. The primary objective of the study was to document any adverse events and establish the safety of the intervention. The secondary objective of the study was to evaluate the effects of the intervention on symptoms of type 1 diabetes, Insulin requirement, blood sugar and HbA1c levels, C peptide levels and autoimmunity.

Participants eligibility criteria and recruitment

Patient selection was based on World Medical Association Helsinki Declaration for Ethical Principles for medical research [3] involving human subjects. A written informed consent was obtained from the parents of all patients. Patients were diagnosed as type 1 diabetes [4] clinically based on following criterion. Most patients had a small age at the time of diabetes with presentation as ketoacidosis and severity of symptoms such as severe weight loss, extreme weakness and with polyuria and bed wetting. On investigations, most of the patients had high sugar levels more than 200 mg/dl with high HbA1c levels. Almost all had very low C peptide levels indicating a very low endogenous Insulin production. Higher than normal Anti-Gad antibody levels were objective evidence of autoimmunity, although was not seen in all patients. All the patients were Insulin dependent since the onset of the disease. The exclusion criteria were presence of acute infections such as HIV/HBV/HCV, malignancies, bleeding tendencies, renal failure, severe liver dysfunction and other acute medical conditions such as respiratory infection and pyrexia.

Preintervention assessment

An informed consent was taken from the parents of all the patients. Prior to intervention, all the patients underwent a thorough clinical examination with serological, biochemical, and hematological tests. Blood sugar fasting and post prandial, Anti Gad antibody titer, Glycosylated Hb and C peptide levels and weight of patient and total Insulin requirement in 24 h were the variables to be measured before the therapy and afterwards at every 3 monthly intervals.

Institutional ethical committee clearance

[5,6] Institutional Ethical Committee (IEC) permission taken, and institutional Committee for Stem Cell Research (IC-SCR) established at city level for peer review of stem cell research and its permission taken for the study. Special informed consent was taken from all patients/relatives.

Patient symptomatology

In the therapy group, 5 patients (23.8%) presented with significant weakness, 15 patients (71.4%) had significant weight loss, 7 patients (33.33%) had polyuria, 6 patients (28.57%) had polyphagia, 3 patients had blurred vision with vision loss (14.2%),3 patients (14.2%) had peripheral neuropathy and 2 patients (9.5%) complained of erectile dysfunction. 3 patients (14.2%) had presented as diabetic ketoacidosis with coma. Only 6 patients (28.5%) had auto immunity with significantly raised Anti Gad antibody levels.

Bone marrow harvesting

Patients below 18 years were given general anesthesia. Whereas patients above 18 years, spinal anesthesia was given. 100–150 ml marrow was aspirated using a Jamshidi bone marrow aspiration needle attached to a disposable 50 ml syringe with heparin sodium as anticoagulants from posterior inferior iliac spine.

Separation protocol for isolation of stem cells

[7] The collected bone marrow e.g., 25 ml is diluted with equal quantity of Dulbecco's Phosphate Buffered Saline with 2% Fetal Bovine Serum (PBS + 2% FBS) (Sigma-Aldrich, USA). The diluted bone marrow is poured over equal quantity of density gradient such as Lymph prep (Stem cell technologies, Vancouver, Canada) with density of 1.077 g/mL into disposable centrifuge tubes of 50 ml capacity. The tubes are centrifuged at 800×g for 20 min at room temperature (15 °C - 25 °C) with brake of. The tubes have a buffy coat at the middle containing mononuclear cells with stem cells. The buffy coat is aspirated and kept ready for infusion. A sample of this was sent for cytology to measure stem cell viability by Trypan blue and to measure stem cell count. The mononuclear cells were checked for viability (average viability count was found to be 97.3%. The average numbers of cells harvested were 7.86 × 107.

The technique of the omental pouch stem cell operation

A small incision is taken within the umbilicus and the sheath cut open to open peritoneal cavity. Omentum identified and taken out of the umbilical incision. Omental pouch was created by making a sack of the Omentum with continuous interlocking sutures of 3-0 Vicryl sutures. No. 7 infant feeding tube (IFT) is placed into it the Omental pouch (Figure: 1) and the tube fixed with a purse string suture of 3-0 Vicryl. Another no. 10 IFT is put into the peritoneal cavity. The omental pouch was placed inside peritoneal cavity and the sheath sutured with 1-0 Proline interrupted stitches.

Mode of transplantation of stem cells

An infusion of 20 mg/kg methylprednisolone in 100 ML normal saline started intravenously over 1 h before transplanting stem cells. The average numbers of cells transplanted were 7.86 × 107. One third quantity of isolated stem cells were put into the Omental pouch through no. 7 IFT, another one third into peritoneal cavity through no. 10 IFT and remaining third is given IV in 100 ml normal saline.

Post stem cell transplantation therapy

Patients were examined regularly for any adverse effects of the therapy. The vitals such as pulse, blood pressure, respiration monitored regularly. Blood sugar fasting and post prandial was regularly monitored by glucometer and a daily chart maintained.

Outcome measures

To assess the safety of the intervention, outcome measures were used to monitor any major or minor adverse events through the entire duration of follow up. Patients were counseled regarding the probable adverse events during informed consent. Recording of adverse events during the hospital stay was done by a health professional whereas after discharging it was recorded, as reported by the parents or primary care givers and patients diabetologists in their local area.

Monitoring procedure related adverse events

Acute procedural adverse events, associated with cell aspiration and injection, were stringently monitored over 5–7 days after intervention. Body temperature, blood sugar fasting and post prandial, blood pressure, respiratory rate, and heart rate were recorded at regular intervals. Aspiration and transplantation sites were examined every day for pain, bleeding, and signs of infection. Signs and symptoms of any anesthesia complications, headache, nausea, and vomiting were checked regularly. All the minor acute procedural adverse events were treated medically prior to the discharge of the patients from the hospital.

Monitoring cellular transplantation related adverse events

During the stay in the hospital, signs and symptoms of any allergic reaction were monitored at regular intervals. Long term major and minor adverse events were monitored to establish the safety of stem cell transplantation.

Monitoring the effects after intervention

The sugar levels were regularly monitored daily by glucometer and the dose of insulin adjusted accordingly. Blood sugar fasting and post prandial, Anti Gad antibody titer, Glycosylated Hb and C peptide levels, patient's weight and total daily Insulin requirement were the variables to be compared. These parameters were checked before the therapy and afterwards at every 3 monthly intervals.

Statistical analysis

The demographic data for all the patients was recorded and analyzed. Mean age in years at the time of intervention, mean age in years at the time of diagnosis, and mean time duration in months at which the patients were followed up were calculated. The pre- and postintervention scores predetermined level of significance at 0.05 and 0.01. Percentage analysis was conducted for the blood sugar fasting and post prandial, Anti Gad antibody titer, Glycosylated Hb and C peptide levels, patient's weight, and total daily Insulin requirement before and after one year of the therapy in both groups. The whole statistical analysis was done in Microsoft Excel program. Statistical hypothesis testing was performed to prove statistical significance to check if the results are better in therapy group by sampling error and by mere chance or by its own merit, thus rejecting the null hypothesis (see Table 1).

Table 1

Patient data table.

Sr. No	Date of therapy	Age (Yr)	Sex	History	Clinical features	InvestigationsBefore therapy	Insulin before therapy (IU)In 24 h	Side effects of therapy	Clinical Improvement1-year after therapy	Units of Insulin after therapy in 24 h					Investigations After therapy
										6 mt	1 yr	2 yr	3 yr	4 yr	6 mt	1 yr	2 yr	3 yr	4 yr
1	August 04, 2017	20	M	KCO type 1 diabetes for 2 years	Weakness, Peripheral neuropathyWeight 51 kg	Hba1c 9.1Blood Sugar F 210Blood Sugar pp 320C peptide 0.05Anti-Gad antibody 23.7	45	Skin rash	Weight gain of 9 kg in 1-year, peripheral neuropathy symptoms disappeared completely	30	20	20	20	20	Hba1c 8.1Blood Sugar F 186Blood Sugar pp 210	Hba1c 7.3Blood Sugar F 156Blood Sugar pp 185C peptide 0.15Anti-Gad antibody 11.6	Hba1c 7.4Blood Sugar F 162Blood Sugar pp 190	Hba1c 7.3Blood Sugar F 152Blood Sugar pp 175	Hba1c 7.4Blood Sugar F 160Blood Sugar pp 185
2	October 13, 2017	15	M	KCO type 1 diabetes for 1 year	Weakness, Bed wetting, Peripheral neuropathy,Weight 53 kg	Hba1c 12.4Blood Sugar F 310Blood Sugar pp 452C peptide 0.01Anti-Gad antibody 13.7	70	nil	Weight gain of 7 kg in 1-year, peripheral neuropathy symptoms disappeared completely, bed wetting completely stopped	50	35	35	35	35	Hba1c 8.3Blood Sugar F 185Blood Sugar pp 258C peptide 0.15	Hba1c 8.1Blood Sugar F 180Blood Sugar pp 252C peptide 0.15Anti-Gad antibody 13.7	Hba1c 8.2Blood Sugar F 175Blood Sugar pp 252	Hba1c 8.1Blood Sugar F 170Blood Sugar pp 248	Hba1c 8.2Blood Sugar F 175Blood Sugar pp 262
3	November 29, 2017	5	M	KCO type 1 diabetes for 6 months	HO of diabetic ketoacidosis with coma, polydipsia, Weight 16 Kg	Hba1c 9.5Blood Sugar F 226Blood Sugar pp 358C peptide 0.5Anti-Gad antibody 5.47	12	Pain in abdomen	Weight gain of 4 kg in 1-year, improved energy, polydipsia disappeared completely	6	0	0	0	0	Hba1c 5.3Blood Sugar F 91Blood Sugar pp 98C peptide 0.58	Hba1c 5.0Blood Sugar F 73Blood Sugar pp 78C peptide 0.98	Hba1c 5.1Blood Sugar F 89Blood Sugar pp 92	Hba1c 5.2Blood Sugar F 94Blood Sugar pp 98	Hba1c 5.0Blood Sugar F 78Blood Sugar pp 82
4	25/8/2018	20	M	KCO type 1 diabetes for 1 year	HO extreme weight loss and polyuria, erectile dysfunction,Weight 37 Kg	Hba1c 14Blood Sugar F 292Blood Sugar pp 358C peptide 0.1Anti-Gad antibody 5.47	55	nil	Weight gain of 13 kg in 1-year, Improved energy, improvement in erectile dysfunction	25	25	25	25	NA	Hba1c 7.8Blood Sugar F 201Blood Sugar pp 213C peptide 0.3	Hba1c 7.5Blood Sugar F 193Blood Sugar pp 203C peptide 0.42	Hba1c 7.3Blood Sugar F 186Blood Sugar pp 201	Hba1c 7.4Blood Sugar F 176Blood Sugar pp 198	NA
5	December 10, 2018	27	M	KCO type 1 diabetes for 5 years	HO extreme weight loss and polyuria, Weight 68 Kg	Hba1c 11.8Blood Sugar F 250Blood Sugar pp 358C peptide 0.05Anti-Gad antibody 3.58	40	Skin rash	Weight gain of 3 kg in 1-year, no polyuria	25	20	20	20	NA	Hba1c 7.5Blood Sugar F 169Blood Sugar pp 230C peptide 0.10	Hba1c 6.5Blood Sugar F 140Blood Sugar pp 210C peptide 0.15	Hba1c 6.8Blood Sugar F 169Blood Sugar pp 230	Hba1c 7.0Blood Sugar F 154Blood Sugar pp 215	NA
6	December 11, 2018	21	F	KCO type 1 diabetes for 15 years	HO weight loss and polyuria, weakness, Peripheral neuropathy,Weight 58 Kg	Hba1c 11.5Blood Sugar F 250Blood Sugar pp 350C peptide 0.22Anti-Gad antibody 5.47	50	nil	Weight gain of 8 kg in 1-year, Improved energy, no polyuria	25	25	25	25	NA	Hba1c 7.3Blood Sugar F 150Blood Sugar pp 220C peptide 0.36	Hba1c 7.0Blood Sugar F 130Blood Sugar pp 190C peptide 0.45	Hba1c 7.3Blood Sugar F 163Blood Sugar pp 201	Hba1c 7.1Blood Sugar F 143Blood Sugar pp 198	NA
7	March 12, 2019	3.5	M	KCO type 1 diabetes for 5 months	HO weight loss and polyuria, HO ketoacidosis,Weight 16 Kg	Hba1c 14Blood Sugar F 355Blood Sugar pp 550C peptide 0.23Anti-Gad antibody 29.77	10	Mild nausea	Weight gain of 4 kg in 1-year, improved energy	5	5	5	5	NA	Hba1c 8.7Blood Sugar F 203Blood Sugar pp 248C peptide 0.23	Hba1c 8.3Blood Sugar F 192Blood Sugar pp 230C peptide 0.56Anti-Gad antibody 19.2	Hba1c 8.1Blood Sugar F 183Blood Sugar pp 215	Hba1c 8.3Blood Sugar F 192Blood Sugar pp 223	NA
8	May 06, 2019	19	M	KCO type 1 diabetes for 7 years	HO weight loss and polyphagiaWeight 50 Kg	Hba1c 5.95Blood Sugar F 126Blood Sugar pp 210C peptide 0.52Anti-Gad antibody 5.2	42	nil	Weight gain of 10 kg in 1-year, Improved energy, no polyphagia	25	15	15	NA	NA	Hba1c 4.8Blood Sugar F 96Blood Sugar pp 154C peptide 0.55	Hba1c 4.4Blood Sugar F 90Blood Sugar pp 140C peptide 0.55	Hba1c 4.0Blood Sugar F 80Blood Sugar pp 110C peptide 0.75	NA	NA
9	June 24, 2019	11	M	KCO type 1 diabetes for 1years	HO weight loss and polyphagiaWeight 38 Kg	Hba1c 9.4Blood Sugar F 239Blood Sugar pp 378C peptide 0.23Anti-Gad antibody 98	28	Pain in abdomen	Weight gain of 6 kg in 1-year, Improved energy	15	30	30	NA	NA	Hba1c 7.4Blood Sugar F 166Blood Sugar pp 248C peptide 0.22	Hba1c 6.62Blood Sugar F 143Blood Sugar pp 228C peptide 0.42Anti-Gad antibody 55	Hba1c 6.58Blood Sugar F 141Blood Sugar pp 221	NA	NA
10	August 30, 2019	21	M	KCO type 1 diabetes for 2 years	HO stem cell therapy 1 year ago,Weight 50 Kg	Hba1c 7.5Blood Sugar F 193Blood Sugar pp 203C peptide 0.42	25	nil	Weight gain of 3 kg in 1-year, Improved energy	10	0	0	NA	NA	Hba1c 6.1Blood Sugar F 128Blood Sugar pp 168C peptide 0.35	Hba1c 5.5Blood Sugar F 111Blood Sugar pp 153C peptide 0.65	Hba1c 5.8Blood Sugar F 121Blood Sugar pp 161	NA	NA
11	September 04, 2019	14	M	KCO type 1 diabetes for 7 years	HO weight loss and polyphagia, bed wetting,Weight 32 Kg	Hba1c 10.5Blood Sugar F 238Blood Sugar pp 339C peptide 0.02Anti-Gad antibody 42.2	45	Pain in abdomen	Weight gain of 5 kg in 1-year, Improved energy, no bedwetting	45	25	25	NA	NA	Hba1c 8.9Blood Sugar F 201Blood Sugar pp 231C peptide 0.1	Hba1c 8.5Blood Sugar F 195Blood Sugar pp 211C peptide 0.3Anti-Gad antibody 18	Hba1c 8.4Blood Sugar F 189Blood Sugar pp 209	NA	NA
12	September 06, 2019	25	M	KCO type 1 diabetes for 7 years	HO weight loss and polyphagia, erectile dysfunctionWeight 52 Kg	Hba1c 9.4Blood Sugar F 205Blood Sugar pp 278C peptide 0.05Anti-Gad antibody 17.5	50	nil	Weight gain of 8 kg in 1-year, Improved energy, improvement in erectile dysfunction, no polyphagia	30	20	20	NA	NA	Hba1c 8.1Blood Sugar F 167Blood Sugar pp 215C peptide 0.08	Hba1c 7.8Blood Sugar F 158Blood Sugar pp 205C peptide 0.1	Hba1c 8.0Blood Sugar F 178Blood Sugar pp 215	NA	NA
13	December 14, 2019	10	M	KCO type 1 diabetes for 6 months	HO weight loss and polyuriaWeight 38 Kg	Hba1c 11.4Blood Sugar F 250Blood Sugar pp 410C peptide 0.15Anti-Gad antibody 2000	18	Mild Skin rash	Weight gain of 5 kg in 1-year, Improved energy, no polyuria	10	20	20	NA	NA	Hba1c 9.5Blood Sugar F 226Blood Sugar pp 221C peptide 0.15	Hba1c 8.9Blood Sugar F 209Blood Sugar pp 210C peptide 0.35Anti-Gad antibody 950	Hba1c 9.1Blood Sugar F 215Blood Sugar pp 231	NA	NA
14	December 23, 2019	38	M	KCO type 1 diabetes for 5 years	HO weight loss and polyphagia,Weight 58 Kg	Hba1c 10.4Blood Sugar F 252Blood Sugar pp 358C peptide 0.05Anti-Gad antibody 1.3	50	nil	Weight gain of 3 kg in 1-year, improved energy, no polyuria	40	25	25	NA	NA	Hba1c 8.8Blood Sugar F 206Blood Sugar pp 238C peptide 0.8	Hba1c 8.4Blood Sugar F194Blood Sugar pp 221C peptide 0.15	Hba1c 8.6Blood Sugar F198Blood Sugar pp 228	NA	NA
15	December 28, 2019	29	M	KCO type 1 diabetes for 8years	HO weight loss and polyuria, blurred vision,Weight 55 Kg	Hba1c 11.6Blood Sugar F 286Blood Sugar pp 368C peptide 0.15Anti-Gad antibody 17	45	Pain in abdomen	Weight gain of 6 kg in 1-year, improved energy, no polyuria, improved vision		25		NA	NA	Hba1c 9.1Blood Sugar F 214Blood Sugar pp 248C peptide 0.15	Hba1c 8.6Blood Sugar F 200Blood Sugar pp 231C peptide 0.35	Hba1c 8.5Blood Sugar F 196Blood Sugar pp 221	NA	NA
16	February 26, 2020	5	M	KCO type 1 diabetes for 6 months	HO of diabetic ketoacidosis with coma, HO weakness and polyuriaWeight 18 Kg	Hba1c 9.8Blood Sugar F 235Blood Sugar pp 358C peptide 0.14Anti-Gad antibody 2000	20	Nil	Weight gain of 1 kg in 1-year, improved energy	10	20	NA	NA	NA	Hba1c 11.6Blood Sugar F 286Blood Sugar pp 395C peptide 0.12	Hba1c 10.8Blood Sugar F 265Blood Sugar pp 385C peptide 0.24Anti-Gad antibody 830	NA	NA	NA
17	October 14, 2020	40	M	KCO type 1 diabetes for 15 years	HO weight loss, lack of energy and polyphagiaWeight 50 Kg	Hba1c 7.2Blood Sugar F 128Blood Sugar pp 174C peptide 0.3Anti-Gad antibody 12.67	38	Mild nausea	Weight gain of 9 kg in 1-year, Improved energy, no polyphagia	25	15	NA	NA	NA	Hba1c 7.1Blood Sugar F 157Blood Sugar pp 158C peptide 0.35	Hba1c 6.2Blood Sugar F 101Blood Sugar pp 138C peptide 0.65Anti-Gad antibody 12.67	NA	NA	NA
18	October 19, 2020	17	F	KCO type 1 diabetes for 2 years	HO weight loss, lack of energy Weight 48 Kg	Hba1c 16.8Blood Sugar F 256Blood Sugar pp 333C peptide 0.18Anti-Gad antibody 7.4	45	nil	Weight gain of 10 kg in 1-year, Improved energy	25	25	NA	NA	NA	Hba1c 10.9Blood Sugar F 209Blood Sugar pp 258C peptide 0.18	Hba1c 8.5Blood Sugar F 158Blood Sugar pp 238C peptide 0.28	NA	NA	NA
19	January 12, 2021	24	M	KCO type 1 diabetes for 3years	HO weight loss, HO vision loss, blurred vision, Weight 58 Kg	Hba1c 12.2Blood Sugar F 195Blood Sugar pp 394C peptide 0.25Anti-Gad antibody 1.2	36	nil	Weight gain of 8 kg in 6 months, improvement in vision	18	0	NA	NA	NA	Hba1c 8.8Blood Sugar F 175Blood Sugar pp 255C peptide 0.46	Hba1c 8.5Blood Sugar F 150Blood Sugar pp 245C peptide 0.98	NA	NA	NA
20	May 12, 2021	13	M	KCO type 1 diabetes for 12years	HO of diabetic ketoacidosis with coma, Dwarfism, inability to walk due to hip arthritisWeight 8.6 kg	Hba1c 12.95Blood Sugar F 312Blood Sugar pp 394C peptide 0.25Anti-Gad antibody 9.14	20	Pain in abdomen	Weight gain of 2 kg in 6 months	10	NA	NA	NA	NA	Hba1c 9.3Blood Sugar F 155Blood Sugar pp 210C peptide 0.25Anti-Gad antibody 9.14C peptide 0.55	NA	NA	NA	NA
21	May 18, 2021	37	M	KCO type 1 diabetes for 2 years	HO weight loss, HO vision loss, Weight 48 Kg	Hba1c 12.4Blood Sugar F 295Blood Sugar pp 380C peptide 0.02Anti-Gad antibody 9.14	36	nil	Weight gain of 10 kg in 6 months	25	NA	NA	NA	NA	Hba1c 8.4Blood Sugar F 195Blood Sugar pp 210C peptide 0.15	NA	NA	NA	NA

NA= Not Applicable.

Results

The minimum follow up was 6 months and maximum of 4 years. It took 3 months to see the results. The sugar levels were regularly monitored daily by glucometer by the parents and the dose of insulin adjusted accordingly by the patient's physician. Blood sugar fasting and post prandial, Anti Gad antibody titer, Glycosylated Hb and C peptide levels, patient's weight and total daily Insulin requirement were the variables to be compared. These parameters were checked before the therapy and afterwards at every 3 monthly intervals. The clinical results were completely expressed at the end of one year from the therapy. Hence, the above variables were compared at the end of one year from the therapy date (Table 1). Some patients did not send investigation reports at 3 monthly intervals due to variety of reasons such as lack of affordability, lack of motivation, lack of availability in their local area of residence etc. Weight gain is an important prognostic indicator in type 1 diabetes. Weight just before therapy and one year after therapy was measured in both groups. In the therapy group, the average weight gain after therapy was about 16% which came as statistically highly significant with P < 0.01 (Chart A). In the control group, the average weight gain was just 2.23% and the difference was not statistically significant (Chart B).

Chart A and B

showing average and percentage weight gain in both groups.

At the end of one year, in the therapy group, 3 (14.28%) patients went off Insulin and are free of insulin till three years after therapy. Remaining 19 (85.72%) patients average insulin requirements dropped by 51.68% after one year of therapy and the difference came as statistically very significant with P < 0.01 (Chart C). In the control group, no patient went off Insulin and only 13.2% patient's Insulin requirements were lower than before, and the difference was not significant statistically (Chart D). 58.6% patients Insulin requirements were the same and 28.2% patients Insulin requirements were higher than before.

Chart C and D

showing a drop in average daily Insulin requirement in therapy and control group.

HbA1c levels has an average drop of 35.78% drop in the therapy group and the difference came as statistically very significant with P < 0.01 (Chart E). The control group had an average drop of only 8.2% and the difference was not statistically significant (Chart F), 62.6% patients had same levels and 29.2% patients had levels higher than before.

Chart E and F

showing average drop of HbA1c levels in therapy and control group.

There was an average drop of 42.8% in fasting blood sugar in therapy group and the difference was statistically highly significant with P < 0.01 (Chart G). In the control group, there was an average drop of 8.1% in fasting blood sugar levels and the difference was not statistically significant (Chart H). 59.3% patients had the same fasting blood sugar, and 32.6% patients had a higher post prandial blood sugar level.

Chart G and H

showing average drop of fasting blood sugar levels in therapy and control group.

There was an average drop of 50.93% in post prandial blood sugar in therapy group and the difference was statistically highly significant with P < 0.01 (Chart I). In the control group, there was an average drop of 13.1% in post prandial blood sugar levels and the difference was not statistically significant (Chart J). 5.3% patients had the same post prandial blood sugar, and 31.6% patients had a higher post prandial blood sugar level.

Chart I and J

showing average drop of post prandial blood sugar levels in therapy and control group.

There was an average increase of 89% C peptide levels in therapy group and the difference was statistically highly significant with P < 0.01 (Chart K). In the control group, there was an average increase of 11.1% in C peptide levels and the difference was not statistically significant (Chart L). 65.1% patients had the same C peptide levels, and 23.8% patients had a higher C peptide level.

Chart K and L

showing average drop of C peptide levels in therapy and control group.

In the therapy group, 6 patients had a higher-than-normal Anti-GAD antibody titer which dropped to about 67% in one year indicating partial reversal of auto immunity (Chart M). In the control group, 11 patients had a higher-than-normal Anti-GAD antibody titer and only 13.8% of them had a lower level at the end of one year (Chart N).

Chart M and N

showing average drop of Anti GAD antibody levels in therapy and control group.

There were a few side effects noted after stem cell therapy, but they were largely self-limiting and minor in nature. 3 patients (14.2%) had a mild skin rash which disappeared without any treatment. 5 patients (23.8%) had post-operative pain which completely resolved with a single dose of Diclofenac Na injection as an analgesic. 2 patients (9.5%) had mild nausea which did not require any treatment.

Discussion

Adult stem cells are of 2 types [8], mesenchymal and haemopoietic stem cells. The doubling time is only 15.8 h [8]. They have potential to duplicate indefinitely and differentiate into 22 types of cells [9]. These cells typically divide for up to 6 months making them into billions of cells. These cells produce 50 types of growth factors and cytokines [11] which repair and differentiate adult tissues in an epigenetic manner. Stem cells put in a particular organ gets differentiated into cells of that organ due to growth factors produced by that tissue. Mesenchymal stem cells can differentiate into tissues of all 3 lineages a phenomenon called plasticity [[12], [13], [14]]. Whenever a particular organ undergoes damage, stem cells in that area come fore-wards and get differentiated into specialized cells and the damage is replenished. But when the damage is extensive, the organ starts failing. By doing stem cell therapy we harvest stem cells from other healthy tissues as fat and bone marrow and put them into the diseased organ and these cells now get differentiated into specialized cells and the organ starts functioning again. In health stem cells from fat or bone marrow can-not migrate into diseased organs, hence it is necessary to transplant them.

Autoimmune markers [15] include autoantibodies to glutamic acid decarboxylase (GAD), insulin, islet cells, islet antigens (IA2 and IA2-beta), and the zinc transporter ZnT8. Since only anti GAD antibody titer was available in my city, hence that was the autoimmune marker used in this series. C peptide levels were used instead of Insulin levels in the study for following reasons. C-peptide and insulin are released from the pancreas at the same time and in about equal amounts. Hence, a C-peptide test [16] can show how much insulin your body is making. This test can be a good way to measure insulin levels because C-peptide tends to stay in the body longer than insulin. C peptide levels are constant than Insulin levels with far less fluctuations.

This Omental pouch stem cell operation (Fig. 1) is reported for the first time in the medical literature. It is based on animal studies and in vitro studies done by other researchers in the past. Stem cells were grown in hyperglycemic environment in a petri dish and after a few weeks developed into Islets like cells that started producing Insulin [17]. These Islets when implanted in diabetic rats reversed diabetes and achieved euglycemia. It is quite likely that the stem cells implanted in the Omental pouch get converted into Islet like cells and produce Insulin due to the hyperglycemic environment of the host. The stem cells acquire blood supply from the surface of Omentum just like a split skin graft which gets its blood supply from granulation tissue. It means that the Omental pouch can act as a new biological pancreas producing Insulin. But we need a separate study to prove it by taking samples of the Omental pouches after one year of therapy and study them under electron microscope to support the claims. Such studies can-not be done in humans as it will raise ethical issues and can reverse the euglycemia achieved by the patient. Following argument is the rationale of placing stem cells into the Omentum. In type 1 diabetes, many patients have auto immunity [18] and implanted Islets of Langerhans are destroyed by the autoimmunity. That makes the treatment of this disease extremely challenging. That is why, a variety of methods have been tried by the researchers in the past such as implanting the Islets into the liver, under kidney capsule, inside the liver, Omentum and into the peritoneal cavity, muscles etc. [19] Placing the stem cells in Omentum saves them from the autoimmunity as cellular immunity on Omental surface is far less than in the blood stream.

Type 1 diabetes is considered an autoimmune disorder. But there were only 6 patients (28.5%) with autoimmunity in my series. It seems that a lot of patients have a one-time damage due to viral infection [20] without auto immunity. These patients had better and long-lasting results than the autoimmunity group as there is no ongoing damage of Islets of Langerhans.

There is evidence today that bone marrow derived mesenchymal stem cells can be transdifferentiated into islet like cells which produce Insulin in Vitro [[21], [22], [23], [24]]. That is the rationale of giving stem cells intravenously. Many of these cells can reach the pancreases and get differentiate into Islet like cells due to specific growth factors released by pancreas. By injecting stem cells intravenously, the autoimmunity can be reversed by the immunomodulatory function of mesenchymal stem cells [25,26].

Mesenchymal stem cells put into peritoneum of rats lead to euglycemia. The MSCs developed into Islet like cells intraperitoneally [27]. The peritoneum has only 10% cellular immunity compared to blood. Above is the rationale of placing one third isolated stem cells into the peritoneum. We already know that stem cells grow till they have 90% confluence levels. It means, stem cells will grow for much longer time in a large space. Peritoneum has a large space; hence stem cells can grow there for a very long time. Since peritoneal surface has far little immunity, the stem cells grow rapidly and for a longer time as there is little immune response against the growth of stem cells. Till the stem cells grow, they produce variety of growth factors and cytokines which are absorbed into blood, and they rejuvenate host tissues including existing Islets of Langerhans.

The therapy group had substantially good results compared to the control group (Table 1). Blood sugar fasting and post prandial, Anti Gad antibody titer, Glycosylated Hb and C peptide levels, patient's weight, and total daily Insulin requirement before and after therapy were the variables to be compared. The difference of above variables was calculated before and after therapy at the end of one year and came statistically highly significant. The therapy was partially successful in reversing auto immunity. Best results were obtained in children without auto immunity. Patients with auto immunity had relatively poor results.

Only one patient repeated the therapy after one year. Stem cells were implanted in peritoneum and blood. He had further 25–30% improvement in the variables compared. Although one patient is not statistically significant, and we need a series with a larger number of patients repeating the therapy and its results. At this stage we can hope that more patients can achieve euglycemia and Insulin independence by repeating the therapy multiple times with a gap of one year.

The study holds a great promise for future research based on above findings. Firstly, it creates hope for a dreadful disease like Type 1 diabetes patients. The allogenic cadaveric Islet cells can be transplanted into an Omental pouch instead of liver. As I have already stated, the Omental surface is best for neo angiogenesis. Hence, the newly transplanted Islets can get vascularized well in the Omental pouch. The Omental pouch surface has far little immunity compared to the blood. Hence, chances of destruction by auto immunity will be far less and the Islets may survive far longer. This study has reported partial reversal of auto immunity in Type 1 diabetes. It's a ray of hope for Type 1 diabetes patients. We need a study specially designed for reversal of auto immunity with large number of patients. The study also reports that only 6 patients (28.5%) out of 21 had auto immunity with significantly raised Anti Gad antibody levels. It supports the alternative hypothesis for type 1 diabetes which states that the disease is due to a one-time viral damage rather than due to auto immunity. If that is true, then regenerating the existing Islets of Langerhans or transplanting new ones makes a sense.

Conclusions

Autologous Stem cell therapy for type 1 diabetes with transplantation of stem cells into the Omental pouch, peritoneum, and blood was safe, and effective for the long term for the treatment of Type 1 diabetes. It also reversed auto immunity partially. We need a greater number of cases and a longer follow up to make it better. The therapy creates a lot of hope for Type 1 diabetes patients as it can be easily repeated any number of times.

Ethical approval

Ethical committee clearance was taken from District IMA Institutional ethical committee and copy attached.

Funding source

The study was self-funded by Dr. Sagar Jawale

Author contribution

Dr. Sagar Jawale is the sole author.

Registration of research studies

Name of the registry: Mendeley Data.

Unique Identifying number or registration ID: 10.17632/2tft8s67dw.2.

Hyperlink to your specific registration (must be publicly accessible and will be checked): https://data.mendeley.com/drafts/2tft8s67dw.

Guarantor

Dr. Ravindra Vora, senior paediatric surgeon, India who is aware of my research Email address: voraravindra@gmail.com.

Consent

Special informed consent was taken from all patients and attached.

Provenance and peer review

Not commissioned, externally peer reviewed.

Declaration of competing interest

I, the only author Dr. Sagar Jawale declare that there is no conflict of interest.