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Manuscript Draft

Manuscript Number: 001.

Title: Combined Treatment of Intrapancreatic Autologous
Bone Marrow Stem Cells and Hyperbaric Oxygen in Type 2
Diabetes Mellitus randomized clinical trial.
Article Type: Original Works or Clinical Submission
Keywords: Bone marrow stem cell, Transplantation,
Hyperbaric Oxygen Therapy, Diabetes.
Esteban J. Estrada , Jose Luis Decima, Camilo Ricordi, Guillermo Bortman, Oscar
Imventarza , Shari Messinger, Elida Beatriz Romero, Maria Julia Esperanza, Gustavo Samaja,
Aldo Rodriguez Saavedra, Eduardo Nicora, Claudio Esteve, Gerardo Martinez, Gustavo
Gimenez, Samuel Gutierrez, Diabetology Department High Complexity Hospital, Diabetology,
Formosa, Argentina.

1- Abstract

2- Background and keywords

3- Introduction

4- Patients and Methods

5- Intervention procedures

6- Outcomes. Statistical considerations and analytical plan

7- Results Tables and graphs

8- Discussion
9- Conclusion
10- References
1- Abstract
T2DM is a pandemic disease with a serious health complications, however stem cells therapy
appear to be a new therapeutic option.

This prospective, open labeled, randomized controlled clinical trial a phase I/II study, comparing
the benefit of hyperbaric oxygen therapy and intra-pancreatic stem cell infusion vs. control group
with standard medical treatment (insulin + metformin) for type 2 diabetes mellitus.

The objective of this study was to determine whether intra-pancreatic infusion of bone marrow
derived autologous stem cells in combination with hyperbaric oxygen therapy could improve
metabolism in patients with type 2 diabetes mellitus. The primary endpoint was HbA1c reduction.

The Patients with T2DM enrolled were 23. They were randomized and assigned, to intervention
group 13 patients and 10 to control group. They were followed for 1 year.

Metabolic variables included (fasting plasma glucose, C-peptide, HbA1c, and calculation of C-
peptide/glucose ratio) and clinical variables (BMI, insulin requirement) were measured at
baseline and at 180, 270 and 365 days.

At baseline there was no significance difference between groups in HbA1C, glucose, c-peptide, c-
peptide/glucose and insulin, BMI was greater in the intervention group than in the control group
(diff=3.93, p=0.006). At 365 days in the intervention group HbA1C changes were significant
(diff=-0.76, p=0.05), glucose (diff=-64.1, diff 0.001), c-peptide (diff=1.25, p< 0.001), c
peptide/glucose (diff=0.01, p< 0.0001). Changes in A1c compared (both groups) to time 0 were
not significant to the control group at any time during follow up , but were significantly decreased
in the intervention group at 365 days (diff=-1.08, p<0.001).

The conclusion of this study was that combination of autologous stem cell therapy and hyperbaric
oxygen treatment could favor tissue remodeling and regeneration- expansion of insulin producing
cells in the treated patients vs. patients with standard medical treatment Insulin and metformin in
a randomized clinical trial.

2- Key Words:

Bone marrow stem cells, Transplantation, Randomized, Hyperbaric Oxygen Therapy, Diabetes
Mellitus type 2.
3- Introduction:
Diabetes mellitus or type 2 diabetes is a chronic disease characterized by a deficit in B-cell mass
and a failure of glucose homeostasis characterized by high levels of glucose in the blood
(hyperglycemia) due to cellular resistance to the actions of insulin, combined with poor drainage
of insulin by the pancreas, resulting in a variety of severe complications and an overall shortened
life expectancy. Insulin deficiency leads to an increase in the level of glucose in the blood, which
itself causes symptoms and may be life-threatening and, in the long term, is associated with
damage to blood vessels and nerves (1).. Patient may have more insulin resistance, while another
may have a major defect in the secretion of the hormone .About 30% or more of patients with
type 2 diabetes are benefited with insulin therapy to control glucose levels in blood.
Type 2 diabetes is the most common in the diabetes mellitus and the difference with type 1
diabetes mellitus it is characterized by autoimmune destruction of insulin-secreting cells by forcing
patients to rely on exogenous insulin for survival, although the most accepted treatment for
diabetes patients requires a rigorous and invasive regimen of repeated testing of blood sugar
levels and injections of recombinant insulin to make up for their own lack of insulin production.
However, even the most effective forms of recombinant insulin and advanced blood glucose
detection systems do not ensure blood glucose control and prevention of long-term complications.
It has also been shown that patients with higher serum levels of C-peptide at the time of diagnosis
suffered less microvascular complications and less hypoglycemic events than those patients with
low levels of C-peptide. Type 2 diabetes is a common and under diagnosed condition that poses
challenges for treatment. The introduction of new drugs orally at the last years has expanded the
range of options available to treat type 2 diabetes but the safety and the reports of complication
attribute for they it’s not clear and the patient complication not decrease . Diet combined with
exercise in order to lose weight does significantly improve cellular sensitivity to insulin even before
reaching the ideal weight. It has been shown that exercise and weight loss in diabetic patients and
prediabetic reduces mortality and improving their living conditions. However it’s necessary to
improved new therapeutics for a Type 2 Diabetes, because the incidence in the world it’s increase
constantly and exponentially . Autologous stem cell therapies are an emerging set of therapies
that show promises with a low side effect profile. They are easily accessible and have the
advantage of avoiding histocompatibility issues while maintaining the potential for differentiation
into multiple cell types. Some adult stem cell therapies are already in clinical trials for a variety of
conditions, including Crohn‘s disease (2), myocardial infarction (3) and graft-versus-host disease
(4, 5). New applications of autologous bone marrow transplantation are currently being
developed either to tackle autoimmunity (6, 7) or to induce regeneration in diseases such as
diabetes (8). It is still too early to judge the effectiveness of these therapies, and at this point
there is a need to generate mechanistic data for virtually all of them. The fate of infused cells is
uncertain, as they could either differentiate into the desired tissues or -as preliminary evidence
suggests- simply provide the damaged tissues with trophic signals that may promote their self-
regeneration. The usual directionality of most clinical studies (from bench to bedside) must be
inverted in this particular case: once a therapy proves safe and effective, animal studies must be
designed to investigate how it works. In the setting of diabetes, both embryonic and adult stem
cells have been induced to form insulin producing cells and/or islet like clusters in vitro (9, 10).
There are many physiological scenarios that can induce an alteration in ß-cell mass, pregnancy,
delivery, insulinoma and more pertinently peripheral resistance. It is when this compensatory
mechanism fails that T2DM develops.

It has been shown that islet precursor cells exist within the pancreas. There is ongoing debate as
to whether these originate from ductal cells (11) within the islet (12) or an as yet undefined
location (13) and under the right circumstances can be induced to form ß-cells. In some instance
de-differentiation and re- differentiation into ß-cells has been performed (14). Hence therapeutic
options lie in inducing differentiation of existing stem cells in vivo or providing stem 1 cells to
replace those that are not differentiating adequately. Some studies have suggested that it is not
be a prerequisite that the stem cells 3 are of pancreatic origin (15). Recently, several observations
suggest that multipotential adult stem cells are capable of producing a whole spectrum of cell
types, regardless of whether or not these tissues are derived from same germ layer; highlighting
the opportunity to manipulate stem cells for therapeutic use. Animal studies have shown that
bone marrow derived stem cells are capable of inducing endogenous pancreatic tissue
regeneration in the mouse model of streptozotocin induced diabetes (16). In a similar study,
multipotent stromal cells from human bone marrow infused into mice treated with streptozocin
resulted in higher levels of mouse insulin and an increase in mouse islets and ß-cells. It is not clear
if this was due to cell protection or new cell formation (17). Hyperbaric oxygen has been used for
decades in the treatment of ischemic wounds and decompression sickness. More recently, it has
been shown that hyperbaric oxygen therapy (HOT) increases bone marrow (BM) nitric oxide (NO)
synthase which causes stem cell mobilization/endothelial progenitor cell (EPC) release (18-19). It is
believed that these cells are attracted to sites of inflammation due to local factors (cytokines,
chemokines, etc). As described above, it is well known that in T2DM there is ongoing inflammation
in the pancreas more so in the earlier stages of the disease. We believe that at the site of the
lesion, mobilized stem cells/EPCs will cause angiogenesis and release of factors that will result in
the local differentiation of progenitor cells. Intra-arterial pancreatic stem cell infusion may
increase the local level of stem cells/EPCs resulting in maximization of the effect described above.
This technique has been described in rats (20). Diabetes has also been shown to impair progenitor
cell mobilization (21) another reason why local stem cell infusion may be important, to overcome
this problem. Differentiation into ß-cells will lead to increased C-peptide production and therefore
improved metabolic control as assessed by the measurement of HbA1c. This will be most likely
associated with decreased insulin requirements as well as reduced metformin dosage. Pilot
observations of treatment of patients with type 2 diabetes mellitus using HOT and intrapancreatic
stem cell infusion (section 1.5.1) from one of our collaborators has demonstrated benefit in a
small group of patients. These patients demonstrated improved glycemic control and beta cell
function over the year following the therapy. The purpose of this protocol is to demonstrate these
findings in a prospective, open label, randomized controlled trial.
Combined autologous stem cell (ASC) plus hyperbaric oxygen in Type 2 Diabetes Mellitus was
published Cell Transplantation, Vol. 17, pp. 1295–1304, 2008. The primary objective of this pilot
clinical trial was to evaluate the safety of ASC–HBO combined in patients with T2DM. The study
hypothesis was that combination of autologous stem cell therapy and hyperbaric oxygen
treatment could favor tissue remodeling and regeneration- expansion of insulin producing cells in
the treated patients.

4- Patients and methods

Study Design
Clinical Phase I/II, prospective, open labeled, randomized controlled clinical trial comparing the
benefit of hyperbaric oxygen therapy, intrapancreatic stem cell infusion or both to standard
medical treatment alone for type 2 diabetes mellitus. 23 patients at baseline, suffering from T2DM
(duration 5-15 years) were randomized to treatment in two groups (13 HBO-ASC, 10 SMT)
.Patients were enrolled December 2009 and the randomization simple was done March 2010 the
follow up was done till March 2011 In the Hospital de Alta Complejidad, Formosa, Argentina.
Patients underwent a combined intervention - Intrapancreatic autologous stem cells infusion +
hyperbaric oxygen sessions before and after the stem cell infusion. Patients provided signed
informed consent before participating local institutional review board and the CUCAIFOR
(regulator entity).

Inclusion Criteria
Patients who meet all of the following criteria are eligible for participation in the study:
1. Male and female patient’s age 45 to 65 years of age.
2. Ability to provide written informed consent.
3. Mentally stable and able to comply with the procedure.
4. Clinical history compatible with type 2 diabetes (T2DM) as defined by the Expert Committee on
the Diagnosis and classification of Diabetes Mellitus (ref 40).
5. Onset of T2DM disease at ≥ 40 years of age.
6. T2DM duration ≥ 5 and ≤ 15 years at the time of enrollment.
7. Basal C-peptide 0.5-2.0 ng/mL
8. HbA1c ≥ 7.5 and ≤ 11% before standard medical therapy (SMT). Patients must have been
treated with SMT for minimum of 4 months prior to randomization. Insulin dose and metformin
doses should be stable over the 3 months prior to randomization.
9. HbA1c ≥ 7.5 and ≤ 9.5% at time of randomization.
10. Total insulin daily dose (TDD) at time of randomization should not exceed 100 units/day
Exclusion Criteria
Patients who meet any of these criteria are not eligible for participation in the study:
1. BMI >35 kg/m2.
2. Insulin requirements of > 100 U/day.
3. HbA1c >9.5%. (at the time of randomization)

Treatment description
Subjects will receive standard medical treatment (SMT) with insulin and metformin for 4 months
(evaluation phase). Then they will be randomized into 2 groups (intervention and control group)

1) Control group: continue with SMT.

2) HOT+SC group: Combination of hyperbaric oxygen therapy and intra pancreatic autologous
stem cell infusion (intervention group) in addition to SMT

Primary Endpoint
The reduction in HbA1c from time of randomization to 1 year after intervention.

Secondary Endpoints
Efficacy Endpoints At 6 months (182 days) ± 14 days following the intervention:

The proportion of subjects with a reduction of ≥1% in HbA1c

The proportion of individuals with HbA1c ≤6.5%
The proportion of subjects off insulin with an HbA1c<6.5%
The percent reduction in insulin requirements the reduction in/cessation of oral hypoglycemic
5- Intervention procedures:
Hyperbaric Oxygen Treatment (HOT)
Hyperbaric oxygen treatment will be administered over a period of 3 weeks according to
protocol standard operating procedure. Briefly, each session of Hyperbaric Oxygen
Therapy (HOT) will be administered in a hyperbaric chamber at a target pressure of 2.3
ATA (absolute atmospheres) with a minimum pressure of 2.0 ATA, at 100% FiO2 for 45
minutes (the time needed to reach the target pressure and decrease it again to normal
pressure is not included). In the combined HOT-SC intervention group there will be 10
sessions before the intra- pancreatic infusion of autologous stem cells and 10 sessions
after. Subjects will equalize pressure in the middle ear using diving techniques, to
minimize discomfort and adverse events. . The stem cell infusion will be scheduled on a
Wednesday to maximize HOT treatments immediately before and after. HOT treatments
will be performed Monday through Saturday over a 3 week period. All subjects will start
vitamin C total daily dose of 1000mg and vitamin E 24 200mg once daily at final visit prior
to randomization This will be continued until the 3 month post intervention visit. The
purpose of this will be an antioxidant effect to minimize potential adverse effects of HOT
secondary to free oxygen radicals.

Rationale for the use of hyperbaric oxygen therapy for the treatment of
patients with diabetes mellitus
There is an increasing body of data in the medical literature describing beneficial effects of
hyperbaric oxygen therapy (HOT) in several clinical conditions and relevant experimental
models. Even though the mechanisms underlying the properties of HOT are not yet fully
characterized, the data is quite interesting data to justify a rationale of its use as part of
strategies aimed at the treatment of diabetes.

Tissue regeneration
Oxygen levels play important biological functions. The oxygen requirements of different
tissues may vary substantially depending on the functional state.

While the use of HOT has been shown beneficial for the treatment of diabetic ulcers
(namely, resulting in the enhancement of wound healing), the mechanisms underlying this
phenomenon remain largely unknown, and only recently it has been demonstrated that
HOT efficiently mobilizes stem cells from the bone marrow that contribute to the tissue
repair process (22).
Application of HOT has been shown to also enhance the engraftment and the tissue repair
effects of stem cell therapies aimed at inducing tissue repair in experimental models of
infarct heart (23) and nerve regeneration (24). The use of HOT may also benefit organ
transplantation. Treatment of multi-organ donors after cerebral death may improve or
precondition the organs to make them less susceptible to ischemic injury (25). Treatment
of living adult liver donors (26) or liver transplant recipients (26) with HOT may promote
liver regeneration (27, 28 and 29).

In the case of pancreatic islets, endocrine cell clusters are richly vascularized and
represent ~1% of the pancreas of which they receive ~20% of the blood supply. This
probably reflects the high metabolic rate of endocrine cells and the need to respond
quickly to the metabolic needs. In mice, it has been demonstrated that the appearance of
endocrine cells in the embryonic pancreas coincides with the formation of new blood
vessels, suggesting a key role of oxygen in the maturation of endocrine cells in the
developing pancreas. Indeed, availability of adequate oxygen concentrations in vitro
enhances the maturation of pancreatic endocrine precursors into insulin- and glucagon-
producing cells (30, 31); possibly via the modulation of the hypoxia-inducible factor 1
alpha pathway (32). Based on these premises, HOT may be beneficial in improving
functional beta cell mass and/or regeneration in diabetes. Preliminary data generated at
the Diabetes Research Institute of the University of Miami suggests HOT might induce an
increase in the beta cell mass in mice (Pileggi & Ricordi, unpublished observation). In
addition, mice transplanted with pancreatic islets and treated with HOT displayed
enhanced islet engraftment possibly via the modulation of local inflammation,
improvement of tissue remodeling and vascularization at the implantation site (33, 34).

Modulation of immunity
Experimental evidence indicates that HOT may have an impact on immune cell function.
Administration of HOT to immunocompetent mice results in a significant decrease in the
cellularity of the spleen and thymus, particularly with a reduction of CD4+CD8+ cell
numbers than of CD4+ or CD8+ single positive T cells in the thymus, and more dramatic
reduction of B cells (B220+) than Thy-1+ T cells in the spleen, respectively. Furthermore, in
a murine model prone to autoimmunity (MRL-lpr/lpr mice), HOT resulted in a marked
reduction of cellularity in otherwise enlarged spleens and lymph nodes, with particular
loss of abnormal B220+Thy-1+ cells in spleens, when compared to untreated controls (35).
This data suggests a potential impact on HOT in the treatment of autoimmune diseases.
In a model of graft versus host disease following bone marrow transplantation in lethally
irradiated mice, HOT ameliorates recipients’ survival and is associated with reduced
numbers of CD3+, CD4+, CD8+, CD4+CD11a+, CD4+CD18+, CD8+CD11a+, CD8+CD18+ T
lymphocytes in the spleens of treated animals (20). Interestingly, it has been described
that HOT was the only effective intervention able to treat a case of mutilating and
resistant vasculitis, resulting in reversal of cutaneous lesions, granulation tissue formation
with growth of new skin to cover the previous extensive deficits (36).

Pre-treatment of islets or human fetal pancreata with high oxygen in culture results in
reduced immunogenicity (37) and indefinite survival upon transplantation in allogeneic or
xenogeneic recipients (38, 39). Similar effects have been described in a model of corneal
transplantation where HOT resulted in depletion of Langerhans cells and acceptance of
allogeneic grafts long-term. Furthermore, combinatorial treatment of experimental
animals with cyclosporine and HOT can prevent rejection of allogeneic skin grafts (40, 41
|and 42).

Reported beneficial impact of HOT in type 2 diabetes

Encouraging preliminary data have been reported recently in the scientific literature on
the beneficial effects of HOT in patients with type 2 diabetes in the context of non-
randomized pilot clinical trials.

A pilot trial study performed on 25 patients evaluated the impact of the administration of
HOT (1-hr session in a hyperbaric pressure chamber at a target pressure of 2.3–2.5
atmospheres breathing 100% pure oxygen through a facial mask; total of 10 sessions: five
daily sessions conducted prior to and five after the stem cell infusion) in combination with
intra-arterial injection of bone marrow mononuclear cells obtained from iliac crest
aspirates in patients with type 2 diabetes. The results of this trial suggest that improved
glycemic control and reduced insulin requirements can be achieved in patients with type 2
diabetes using this protocol. Notably, based on these encouraging observations, a
randomized, clinical trial has been designed that involves centers in Argentina (Stem Cell
Argentina, Buenos Aires), USA (Diabetes Research Institute, Miami, FL), and China (Fuzhou
General Hospital, Fuzhou, Fujian). Aim of the studies is to evaluate the impact of HOT and
cellular therapies to restore beta cell function and metabolic control in patients with type
2 diabetes (

The effect of HOT alone on the metabolic control of patients with type 2 diabetes has also
been studied. In a series of 28 patients with type 2 diabetes with diabetic foot ulcers,
exposure to HOT (100% oxygen, 2.4 ATA, 105 min five times a week for 30 sessions) was
associated with significantly improved glycemic control measured as fasting blood glucose
and HbA1c levels, as well as HOMA-IR (43).
Impact of hyperbaric oxygen therapy on type 1 diabetes
At the Diabetes Research Institute of the University of Miami, ongoing studies are
evaluating the impact of HOT in the most stringent preclinical model of type 1 diabetes,
the Non Obese Diabetic (NOD) mouse (45). Preliminary results are showing that HOT can
significantly reduce the incidence of spontaneous or accelerated onset of autoimmune
diabetes in NOD mice, which is paralleled by a significant reduction of insulitis score in
pancreatic sections and by increased beta cell proliferation in the animals undergoing
HOT, when compared to untreated controls (Pileggi & Ricordi, unpublished observation).
These data are encouraging as they indicate that HOT might represent a viable adjuvant
therapy to be included in protocols aimed at halting the progression of autoimmune
diabetes and preventing beta cell loss in the clinical settings.

Hyperbaric oxygen therapy is a safe, non-invasive therapy with virtually absent side
effects. The increasing data supporting the beneficial impact of hyperbaric oxygen
therapy on tissue repair, regeneration and immunity justifies cautious optimism. These
characteristics make of HOT a suitable candidate to further exploration of its possible
clinical applications, including in diabetes prevention trials as single strategy or in
combination with other treatments.

Autologous Stem Cell Infusion (SC)

Bone marrow contains hematopoietic stem cells (HSCs), endothelial progenitor cells
(EPCs) mesenchymal stem cells (MSCs) and recently Vcells (Very Small Embryonic-Like
Stem Cells).
Bone Marrow Harvest Autologous stem cells will be harvested under local and sedation
anesthesia, using multiple bone marrow aspirations from both iliac crests according to
protocol standard operating procedure (appendix 2). Briefly, a minimum of 100 ml and
maximum (target) of 375 ml of bone marrow will be mixed with anticoagulant (heparin)
20,000 units and 1 preservative in a quadruple collection bag (Rivero Brand or similar .If
the target volume of 375 ml could not be achieved, the difference in the volume will be
made up to 375 ml adding peripheral blood to the quadruple blood collection bag. A
minimum of 100 ml of bone marrow is necessary to proceed with the protocol. If this
volume can not be aspirated the patient will be excluded. Exercise is known to facilitate
bone marrow harvest and subjects will be advised to perform a minimum of 45 minutes
walking 3 times a week since enrollment (4 months before harvest) and 3 months after
During the visit on week 13th patients will be reminded about the importance of the
exercise before the harvest in order to improve bone marrow harvest results. Units will
be labeled with the following information:

1. Autologous Donor name

2. Autologous Donor ID
3. Product specification (eg blood/bone marrow)
4. Modifications notes (eg additives – heparin)
5. Source of product (eg iliac crest)
6. Product storage temperature
7. Product total volume
8. Product extraction date and time
9. Autologous Donor Sex
10. Autologous donor blood group and rhesus factor

Bone Marrow Processing

Units will be processed using centrifugation according to the protocol standard operating
procedure (appendix 3). The main objective of this is to separate red cells, plasma and fat
from the buffy coat layer and subsequently get a buffy coat cell count. Briefly, using
centrifugation, gravity flow and the various bags of the quadruple bag system, the red
cells will be discarded in the second bag, the buffy coat collected in the third bag and the
plasma and fat discarded with the first bag. The buffy coat will be washed and
resuspended in isotonic normal saline in the third bag for the final product. This will be
transported for immediate infusion into the pancreatic artery.

Intra-Arterial Pancreatic Autologous Stem Cell Infusion

The infusion of the autologous stem cells will be performed into the main arterial supply
of the pancreas to maximize the presence of stem cells where the therapeutic effect is
most desired. The pancreas typically has several arterial branches that supply arterialized
blood to the organ. The head is supplied by branches of the superior and inferior
pancreatic-duodenal arcade, a network of arterial branches originating from the gastro-
duodenal artery and superior mesenteric artery. The body and tail of the pancreas are
supplied by the dorsal pancreatic artery, a branch of the splenic artery just after its origin
from the celiac axis. Additional small branches arise from the splenic artery as it courses
adjacent to the superior border of the pancreas towards the splenic hilum. Since the
dorsal pancreatic artery supplies the largest volume of pancreatic tissue and is an end
organ vessel without supply to other organs it has been selected for site of infusion. The
procedure will be performed according to the protocol standard operating procedure
briefly; access to this vessel is achieved by a groin puncture into the common femoral
artery. The celiac axis is selected using wire and catheter techniques and sub-selection the
dorsal pancreatic artery performed. Depending on the caliber of this vessel in individual
subjects, an appropriate sized catheter will be used. Positioned will be confirmed with
injection of contrast and stem cell infusion performed.

Hyperbaric Oxygen Treatment and Stem Cell Infusion Combined

HOT will be combined with autologous stem cell infusion. Subjects in this group will
complete the initial 10 sessions of HOT; they will then be admitted to a monitored care
setting and will undergo a bone marrow harvest as detailed above. The harvested cells will
be processed immediately as detailed above and prepared for infusion. The subject will be
transferred to the Interventional Radiology suite where they will undergo an arteriogram
of the pancreas as detailed above, the stem cells will be infused on the same day as the
harvest, and the subject recovered and discharged home. On the next day the subject will
begin the second group of 10 sessions of HOT.

Standard Medical Therapy (SMT)

SMT for patients with diabetes should include management of diabetes itself, as well as
dyslipidemia and hypertension: Diabetes will be managed by the endocrinologist
following standards of care according to the guidelines of the American Diabetes
Association ( that should include diabetes education, medical nutrition therapy and
lifestyle interventions to decrease weight and increase activity (all patients will do 45
minutes of walking or similar exercise 3 times/week since enrollment till 16 months after
harvest) (44). Since the therapy for diabetes will be based on insulin and metformin only,
useful guidelines include the consensus statement from the American Diabetes
Association and the European Association for the Study of Diabetes (45) as well as the
reviews by Nathan DM et al (46) Mooradian AD, et al (47) and Hirsch IB, et al (48). At the
time of enrolling patients in the study diabetes therapy will be assessed. All prohibited
medications will be discontinued. If the patient is not on metformin, the dose will be
initiated as described (49), and will be increased as tolerated to a minimum dose of 1000
mg per day, but preferably 1000 mg twice per day. Metformin will be discontinued 48
hours before and after iodine contrast administration (e.g. intra pancreatic autologous
stem cell infusion). Insulin will be adjusted as necessary to target fasting and pre-prandial
glucose values to 70-130 mg/dl and 2 hour postprandial glucose values to less than 180
mg/dl, and HbA1c values. 7.0% without hypoglycemia. Treatment will be reassessed after
2, 6 and 13 weeks and insulin dosage will be adjusted according to SMBG by fingerstick
glucose values performed a minimum of 8 times in the 2 weeks before the visit and
HbA1c results.
Diabetes will be managed by the endocrinologist and adjustments made to metformin and
insulin glargine every three months throughout study. If a patient was not previously on
insulin then starting dose of insulin glargine will be 10 IU per night to be administered
between 9pm and 12 am. If patient was previously on insulin glargine then previous dose
to be continued. During each three month visit, patient will be instructed to bring fasting
capillary blood glucose values from glucometer for the previous 14 days. During the visit
with the endocrinologist the following adjustments to be made:

1. Average fasting glucose value to be calculated from previous 14 days 1 with a minimum
of eight values required (i.e. add all 14 FPG values and 2 divide by 14 to calculate the
average FPG).
2. If average FPG is between 100-120 mg/dl (5.6-6.7 mmol) then basal 4 insulin glargine
will be increased by 2 IU/night,
3. If average FPG is between 120-140 mg/dl (6.7-7.8 mmol) then increase 6 basal insulin
glargine will be increased by 4 IU/night. 7
4. If average FPG is between 141-180 mg/dl (7.8-10.0 mmol) then increase 8 basal insulin
by will be increased 6 IU/night.
5. If average FPG is greater than 180 (10 mmol) then increase basal insulin glargine by will
be increased 8 IU/night.
6. Exception to this will include: no increase in insulin glargine if a FPG < 12 72 mg/dl was
documented once or more per week during previous two 13 weeks, and decrease in
insulin glargine to be done by 2 IU/night if severe hypoglycemia requiring assistance or
FPG <56 mg/dl was documented.

At 17 weeks if the subject HbA1c is between 7.5 and 9.5% then the patient will be eligible
for randomization into SMT alone or SMT in addition to HOT, SC or 18 HOT+SC. After
randomization and intervention, subjects will be followed every 3 months and
adjustments to SMT will be continued as before. Management of dyslipidemia will be
performed following standards of care according to the guidelines of the American
Diabetes Association (50), as well as the Executive Summary of the Third Report of the
National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation and
Treatment of High Blood Cholesterol in Adults (ATP III) (44) and the European Guidelines
on Cardiovascular Prevention in Clinical Practice (45). Every effort will be made to have all
patients on a statin medication as per standard of care to try to achieve LDL levels <100
mg/dl. In addition, aspirin therapy at 81 mg per day will be prescribed to all patients
unless contraindicated. Aspirin will be discontinued 1 week before the bone marrow
aspiration and the autologous intra-pancreatic stem cell infusion.

Analysis Samples
The goal of this study is to provide strong scientific evidence that HbA1c levels in patients
receiving HOT-SC therapy are reduced enough to justify the risks of the procedure when
compared to standard medical therapy. All efficacy analyses will be based on the
intention-to-treat principle. Once a subject has completed randomization, s/he will be
included in the final analysis Details of all statistical analyses will be given in the formal
statistical analysis plan (SAP).

Study Endpoint Assessment

Primary Endpoint
The primary objective of this investigation is to estimate the reduction in HbA1c from
time of randomization to 1 year after HOT-SC therapy. The primary endpoint is the
difference between HbA1c values measured at randomization and at 1 year post
intervention of HOT-SC therapy. The primary analysis will compute an estimate and a 95%
confidence interval for change in HbA1c levels. The primary endpoint should be available
for all enrolled subjects, as we expect no loss to follow up. An exception will be if a death
occurs or if the subject withdraws consent to be followed in which case HbA1c values
measured at randomization will be used in estimation only at that time point. HbA1c
outcomes will be examined for normality of the distribution and if there is no compelling
evidence of non-normality, change in HbA1c level from randomization to 1 year after HOT-
SC therapy will be analyzed using a paired t-test. If there is compelling evidence of non-
normality, a logarithmic, square root, or other appropriate transformation will be done
before analysis of the data.

We will additionally assess change in HbA1c levels between randomization and 1 year post
therapy comparing the HOT-SC group with SMT. Specifically, we will estimate changes in
HbA1c level in the HOT-SC group and the SMT group and compare the estimates of change
between groups with a two sample t-test assuming normality assumptions hold as
described above and applying appropriate transformations to the data if needed. Power
calculations are illustrated below for the corresponding comparisons assessed with the
one or two sample t-tests described.

Secondary Endpoints
The secondary objectives of this investigation are to estimate the change in measures of
pancreatic function and glycemic control from time of randomization to 1 year completion
after intervention. Additional secondary objectives are to estimate the proportion of
subjects reaching desirable levels for measures of pancreatic function on glycemic control.
For each of the measures of pancreatic function and glycemic control described in 3.1.2,
analysis will initially compute an estimate and a 95% confidence interval for change
between baseline and 1 year post intervention time points.

These endpoints should be available for all enrolled subjects, as we expect no loss to
follow up. An exception will be if a death occurs or if the subject withdraws consent to be
followed in which case values measured at randomization will be used in estimation only
at that time point. Continuous endpoints such as measures of pancreatic function and
glycemic control will be examined for normality of the distribution and if there is no
compelling evidence of non-normality, differences between baseline and 1 year post
intervention will be analyzed using a paired t-test. If there is compelling evidence of non-
normality, a logarithmic, square root, or other appropriate transformation will be done
before analysis of the data.

We will additionally assess change in outcome measures described in 3.1.2 between

randomization and 1 year post therapy comparing the HOT-SC group with SMT.
Specifically, for each endpoint, we will estimate changes in the SMT group and in the HOT-
SC group and compare the estimates of change between groups with two sample t-tests
assuming normality assumptions hold as described above and applying appropriate
transformations to the data if needed.

To assess differences in proportions reaching desirable levels of certain measures of

pancreatic function and glycemic control, (ie. the proportion of patients with HbA1c <= 6.5
at 1 year post intervention), two-sample tests of proportions will be used to assess
differences in proportions between groups.
Sample size and power calculations:

The table below illustrates power to detect differences in HbA1c between randomization and 1
year post HOT-SC therapy assuming a two sided paired t-test, a sample size of 10 subjects a
standard deviation of 1%, and a Type 1 error rate of 0.05. It is shown that there is over 80% power
to detect changes in HbA1c above 0.9% , with over 90% power to detect differences between
baseline and 1 year post HOT-SC exceeding 1%.

In this analysis, we compare A1c, glucose, insulin, cpep and cpep/gluc and BMI across groups both
at time 0 (to make sure groups are comparable at time 0) and at 6, 9 and 12 months (180, 270, and
365 days). We additionally compare changes in A1c comparing 180, 270 and 365 days to time 0 to
see if there are differences between groups in the change in A1c as well as to estimate and the
proportions in each group demonstrating at least a 1 unit decrease in A1c as determined by the
individual changes between the 180, 270, and 365 day time-points and 0. Analyses of continuous
outcomes were performed by mixed model regression incorporating data from all time-points
(>=0) observed on the patients and controls, and appropriately accommodating variances used in
test statistics for the repeated measures nature of the data. The binary outcome capturing a
decrease of at least 1 unit of A1c is analyzed with Fisher’s exact test.

7- Results Tables and graphs

There were 10 patients in the control group, 13 in the intervention group. At time=0, there is no
significant differences between groups in A1c, glucose, cpep, cpep/gluc, and insulin. BMI is
greater in the intervention group than in the control group at time=0 (diff=3.93, p=0.006), and this
difference between groups with respect to BMI remains fairly constant throughout the 365 days of
follow up.
A1c is significantly less in the intervention group at 180 days, (difference=-1.47, p<0.001), 270 days
(difference=-0.85, p=0.03), and borders significance at 365 days (diff= -0.76, p=0.05).

Changes in A1c compared to time 0 were not significant in the control group at any time during
follow up, but were significantly decreased in the intervention group at 180 days (diff=-1.65,
p<0.0001), 270 days (diff=-1.11, p<0.001), and at 365 days (diff=-1.08, p<0.001). The estimated
decrease in A1c from baseline is significantly greater in the intervention group at 180 days (diff=-
1.32 p=0.005), but is not significantly different from control at subsequent time points.
30% (3/10) of patients in the control group and 77% (10/13) of patients in the intervention group
demonstrated a decrease in A1c at 180 days (compared to time=0) of at least 1 unit. This was
compared by Fisher’s exact test, and was found to be a significant difference between groups in
the proportion achieving a 1 unit reduction of A1c in this study over the first 6 months (p=0.02).
This difference in proportions did not maintain statistical significance at subsequent time-points,
although the estimated difference in proportion was relatively constant. At 270 days, these
proportions were 40% (4/10 in control group, and 69% (9/13) in intervention group respectively,
and at 365 days, these proportions were 40% (4/10) and 77% (10/13) respectively.
Glucose is significantly less in the intervention group at 180 days (diff=-64.86, p<0.0001),
at 270 days (diff=-60.15, p<0.001), and at 365 days (diff=-64.1, diff<0.001). However, the
change in glucose from time=0 is not significantly different between the groups at any of
these timepoints. Additionally, at time=0, the intervention group had a lower observed
glucose level (diff=-35.42, p=0.07), although this was not significantly different than the
control group.
C-peptide is significantly greater in the intervention group at 180 days (diff=1.07,
p<0.001), at 270 days (diff=0.95, p<0.001) and at 365 days (diff=1.25, p<0.001).
Cpep/gluc is significantly greater in the intervention group at all follow up time-points.
(diff=0.01, p<0.0001)
It’s greater in the intervention group than in the control group at time=0 (diff=3.93,
p=0.006), and this difference between groups with respect to BMI remains fairly constant
throughout the 365 days of follow up.
Insulin is significantly less in the intervention group at 180 days (diff=-11.55, p=0.04), at
270 days (diff=-13.55, p=0.02), and at 365 days (diff=-12.62, p=0.03).
8- Discussion
This study showed and confirms that the combination of HBO therapy and bone marrow stem cell
infusion could improve the control of plasma glucose the Hb A1C and C-peptide in type 2 diabetic
patients. Our therapy reduced the doses of insulin and HbA1C values.
It is believed that due to local factors (cytokines, chemokines, etc.) these cells are attracted to
inflammated sites and may be important for pancreatic islet repair after injury. Differentiation of
local progenitor cells or bone marrow stem cells into β-cells or the decrease of inflammation in the
pancreas, in our hypothesis, leads to increased c peptide production and therefore, to improved
metabolic control assessed by HbA1c measurement. There has been considerable debate
regarding the origin of neo-cells during normal pancreatic tissue maintenance, the nature of the
regenerating cells after injury, the signals directing their induction and the mechanism(s) by which
they regenerate. However, neogenesis of _ cells in the adult pancreas from non B cell progenitors
has been documented post-natal in experimental rodent models of pancreatic damage (Bonner-
Weir et al., 2004; Holland et al., 2004; Xu et al., 2008).
Our speculation is that this strategy to deliver reparative stem cells intra-arterially and to activate
these cells via systemic in vivo HBO may be targeting more than one crucial reparative step that
counteracts the chronic injuries attacking both the EPC and the islets in the diabetes phenotype.
Furthermore, sound evidence in that regard suggests that HBO has the potential to reverse one of
the three diabetic-related EPC impairments at least (mobilization from bone marrow by NOS
activation). Also, diet, exercise and intensive diabetes measures could be addressed. But we have
experienced that BMI registered no important changes during a 1-year follow-up and this could
not be the reason for the steady outcome registered in connection with this trial over a 1-year

In 2007, Voltarelli JC et al [10] in Brazil first reported the use of autologous non myeloablative
hematopoietic stem cell transplantation (AHST) in newly diagnosed type 1 DM .
They chose 15 newly diagnosed (within 6 weeks) type 1 DM patients. The patients were treated
with AHST after conditioning with cyclophosphamide (200mg/kg) and rabbit antithymocyte
globulin (4.5mg/kg). After a mean time of 18.8-month follow-up, 14 patients became insulin free
continously or transiently, in particular 1 patient was free from insulin for 35 months. 13 of 14
patients maintained HbA1C level less than 7%. In 2009, the team added cases and prolonged mean
follow-up to 29.8 months. 20 of 23 patients became insulin free for periods as long as 4 years with
good glucose control. C-peptide levels increased significantly and maintained for about 3 years
especially in patients continuously free from insulin [11]. AHST could improve the function of
pancreatic β-cell and glucose control in type 1 DM patients.
It the setting of type 2 DM, there were few clinical trials. Bhansali A et al [12] applied
autologous bone marrow-derived stem cell transplantation to treat type 2 DM. In that study stem
cells were injected into gastroduodenal arteries in 10 type 2 DM patients who had the disease for
more than 5 years and were dependent on insulin more than 0.7 U/kg/day at least for 1 year. After
a follow-up for 6 months, 7 patients showed a reduction in insulin requirement by 75% as
compared to baseline, 3 of 7 patients were able to discontinue insulin completely continuously or
transiently. Mean HbA1C reduction was 1%. Both fasting and glucagons-stimulated C-peptide
level were significantly improved. HOMA-B (homeostatic model assessment for β-cell function)
increased significantly while HOMA-IR (homeostatic model assessment for insulin resistance) did
not change significantly
The above clinical trial indicated that autologous BMT may improve pancreatic β-cell function in
type 2 DM which is similar to observation in type 1 DM. Although autologous stem cell
transplantation achieved similar benefits in type 1 and type 2 DM including improvements in
glycemic control and C-peptide levels and reduction of insulin requirements.
Type 2 DM is characterized by decline in β-cell function and worsening of insulin resistance, but
decreasing β-cell mass in type 2 DM was predominantly attributed to hyperglycemia, dyslipidemia,
leptin and cytokines. Transplanted bone marrow cells may have limited capacity to differentiate
into β-cells [31]. So BMCs and/or MSCs infused into type 2 DM patients were not as useful as in
type 1 DM patients. This difference might partly explain the less satisfactory results. But
genetically modified hMSCs may be a potential cell source for cell replacement therapy for

In summary, cell therapy of type 2 DM is an attractive therapeutic strategy that may cure the
disease in future. But autologous bone marrow transplantation can improve glucose control in
Type 2 DM.

Regeneration or replacements of β-cells are two major methods for cell therapy. Induced
pluripotent stem cells, embryonic stem cells, bone marrow-derived MSCs and gene therapy are
still under extensive investigation [32-34].

9- Conclusion

This study validates the initial pilot study and demonstrate in a randomized clinical trial that the
Autologous bone marrow stem cells infusion it s a safe and security option treatment for a
Diabetes Mellitus type 2. There have been no complications observed during bone marrow harvest
or during autologous stem cells infusion. Furthermore, there have been no complications
observed during HBO sessions. Should these benefits persist, diabetes long-term complications
may be avoided, thus decreasing morbidity and mortality associated to the disease.
10- References
1) L. Jorge Goneza,b,∗, Kenneth R. Knight Cell therapy for diabetes: Stem cells, progenitors or beta-
cell replication

2) Garcia-Olmo D, Garcia-Arranz M, Herreros D, Pascual I, Peiro C, 21 Rodriguez-Montes JA. A

phase I clinical trial of the treatment of Crohn's 22 fistula by adipose mesenchymal stem cell
transplantation. Dis Colon 23 Rectum. Jul 2005;48(7):1416-1423.

3) Hare Jea. A Double-Blind, Randomized, Placebo Controlled Clinical Trial of Allogeneic

Mesenchymal Stem Cells for the Treatment of Patients With Acute Myocardial Infarction. Paper
presented at: Innovation in Intervention: 2 Summit, 2007; New Orleans .

4) Fang B, Song YP, Liao LM, Han Q, Zhao RC. Treatment of severe therapy-31 resistant acute graft-
versus-host disease with human adipose tissue-derived 32 mesenchymal stem cells. Bone Marrow
Transplant. Sep 2006;38(5):389-390. 33

5) Ringden O, Uzunel M, Rasmusson I, et al. Mesenchymal stem cells for 35 treatment of therapy-
resistant graft-versus-host disease. Transplantation. 36 May 27 2006;81(10):1390-1397.

6) Voltarelli JC, Couri CE, Stracieri AB, et al. Autologous nonmyeloablative hematopoietic stem cell
transplantation in newly diagnosed type 1 diabetes mellitus. Jama. Apr 11 2007;297(14):1568-

7) Fernández-Viña R, Saslavsky, J., Camozzi, L., Ferreira, J., Andrin, O., Foressi, F., Fernández-Viña,
F., Dadamo, C., Vrsalovic, F. Direct pancreas implant by selective catheterization of spleen artery
of autologous adult mononuclear CD34+/CD38- cells to increase insulin and C-peptide in type I
diabetic patients. Paper presented at: Spring 2007 meeting of the Genetics Society, the British
Society for Developmental Biology and the British Society of Cell Biology, 2007; Heriot-Watt
University, Edinburgh.
8) Baharvand, H.; Jafary, H.; Massumi, M.; Ashtiani, S.K. Generation of 14 insulin-secreting cells
from human embryonic stem cells. Dev Growth 15 Differ. 2006 Jun;(5):323-32.

9) Hori Y, Rulifson IC, Tsai BC, Heit JJ, Cahoy JD, Kim SK. Growth 18 inhibitors promote
differentiation of insulin – producing tissue from 19 embryonic stem cells. Proc Natl Acad Sci U S A.
2002 Dec 20 10;99(25):16105-10. Epub 2002 Nov 19. 21

10) Kim D, Gu Y, Ishii M, Fujimiya M, Qi M, Nakamura N, Yoshikawa T, Sumi 23 S, Inoue K. In vivo

functioning and transplantable mature pancreatic islet-24 like cell clusters differentiated from
embryonic stem cell. Pancreas. 2003 25 Aug;27(2):e34-41. 26

11) Ramiya VK, Maraist M, Arfors KE, Schatz DA, Peck AB, Cornelius JG. 28 Reversal of insulin-
dependent diabetes using islets generated in vitro from 29 pancreatic stem cells. Nat Med. 2000
Mar;6(3):278-82. 30
12) Yang L, Li S, Hatch H, Ahrens K, Cornelius JG, Petersen BE, Peck AB. In 32 vitro trans-
differentiation of adult hepatic stem cells into pancreatic 33 endocrine hormone-producing cells.
Proc Natl Acad Sci U S A. 2002 Jun 34 11;99(12):8078-83. Epub 2002 Jun 4.

13) Hao E, Tyrberg B, Itkin-Ansari P, Lakey JR, Geron I, Monosov EZ, Barcova 37 M, Mercola M,
Levine F. Beta-cell differentiation from nonendocrine 38 epithelial cells of the adult human
pancreas. Nat Med. 2006 Mar;12(3):310-39 6. Epub 2006 Feb 19. 40.

14) Lipsett M, Finegood DT. Beta-cell neogenesis during prolonged 1 hyperglycemia in rats.
Diabetes. 2002 Jun;51(6):1834-41. 2

15) Choi Y, Ta M, Atouf F, Lumelsky N. Adult pancreas generates multipotent 4 stem cells and
pancreatic and nonpancreatic progeny. Stem Cells. 5 2004;22(6):1070-84.

16) Lipsett MA, Castellarin ML, Rosenberg L. Acinar plasticity: development of 8 a novel in vitro
model to study human acinar-to-duct-to-islet differentiation. Pancreas. 2007 May;34(4):452-7.

17) Lee RH, Seo MJ, Reger RL, Spees JL, Pulin AA, Olson SD, Prockop DJ. Multipotent stromal cells
from human marrow home to and promote repair of pancreatic islets and renal glomeruli in
diabetic NOD/scid mice. Proc Natl Acad Sci U S A. 2006 Nov 14;103(46):17438-43. Epub 2006 Nov

18) Goldstein LJ, Gallagher KA, Bauer SM, Bauer RJ, Baireddy V, Liu ZJ, 21 Buerk DG, Thom SR,
Velazquez OC. Endothelial progenitor cell release into circulation is triggered by hyperoxia-
induced increases in bone marrow nitric oxide. Stem Cells. 2006 Oct;24(10):2309-18. Epub 2006
Jun 22. 24. 25

19) Thom SR, Bhopale VM, Velazquez OC, Goldstein LJ, Thom LH, Buerk DG. Stem cell mobilization
by hyperbaric oxygen. Am J Physiol Heart Circ Physiol. 2006 Apr;290(4):H1378-86. Epub 2005 Nov

20) Alvarez SS, Jiménez LM, Murillo AZ, Gómez IG, Ligero JM, Gómez- Pineda A, Rollán-Landeras E,
Cuevas P, Jara-Albarrán A. A new approach for bone marrow-derived stem cells intrapancreatic
autotransplantation in diabetic rats. Microsurgery. 2006;26(7):539-

21) Fadini GP, Sartore S, Schiavon M, Albiero M, Baesso I, Cabrelle A, Agostini C, Avogaro A.
Diabetes impairs progenitor cell mobilisation after hindlimb ischaemia-reperfusion injury in rats.
Diabetologia. 2006 Dec;49(12):3075-84. Epub 2006 Oct 27.

22) Thom SR, Bhopale VM, Velazquez OC, Goldstein LJ, Thom LH, Buerk 9 DG. Stem cell
mobilization by hyperbaric oxygen. Am J Physiol Heart Circ 10 Physiol. 2006 Apr;290(4):H1378-86.
Epub 2005 Nov 18.

23) Khan M, Meduru S, Mohan IK, Kuppusamy ML, Wisel S, Kulkarni A, Rivera BK, Hamlin RL,
Kuppusamy P 2009 Hyperbaric oxygenation enhances transplanted cell graft and functional
recovery in the infarct heart. Journal of molecular and cellular cardiology 47:275-287
24) Pan HC, Chin CS, Yang DY, Ho SP, Chen CJ, Hwang SM, Chang MH, Cheng FC 2009 Human
amniotic fluid mesenchymal stem cells in combination with hyperbaric oxygen augment peripheral
nerve regeneration. Neurochemical research 34:1304-1316.

25) Bayrakci B 2008 Preservation of organs from brain dead donors with hyperbaric oxygen.
Pediatric transplantation 12:506-509.

26) Suehiro T, Shimura T, Okamura K, Okada T, Okada K, Hashimoto S, Mochida Y, Kuwano H,

Saitoh S, Gotoh F 2008 The effect of hyperbaric oxygen treatment on postoperative morbidity of
left lobe donor in living donor adult liver transplantation. Hepato-gastroenterology 55:1014-1019.

27) Franchello A, Ricchiuti A, Maffi L, Romagnoli R, Salizzoni M 2010 Hyperbaric oxygen therapy in
liver transplantation; is its use limited to the management of hepatic artery thrombosis? Transpl
Int 23:e49-50.

28) Muralidharan V, Christophi C 2007 Hyperbaric oxygen therapy and liver transplantation. HPB
(Oxford) 9:174-182.

29) Mori H, Shinohara H, Arakawa Y, Kanemura H, Ikemoto T, Imura S, Morine Y, Ikegami T,

Yoshizumi T, Shimada M 2007 Beneficial effects of hyperbaric oxygen pretreatment on massive
hepatectomy model in rats. Transplantation 84:1656-1661.

30) Fraker CA, Ricordi C, Inverardi L, Dominguez-Bendala J 2009 Oxygen: a master regulator of
pancreatic development? Biology of the Cell 101:431-440

31) Fraker CA, Alvarez S, Papadopoulos P, Giraldo J, Gu WY, Ricordi C, Inverardi L, Dominguez-
Bendala J 2007 Enhanced oxygenation promotes beta-cell differentiation in vitro. Stem cells
(Dayton, Ohio) 25:3155-3164.

32) Heinis M, Simon MT, Ilc K, Mazure NM, Pouyssegur J, Scharfmann R, Duvillie B Oxygen tension
regulates pancreatic beta-cell differentiation through hypoxia-inducible factor 1alpha. Diabetes

33) Juang JH, Hsu BR, Kuo CH, Uengt SW 2002 Beneficial effects of hyperbaric oxygen therapy on
islet transplantation. Cell transplantation 11:95-101.

34) Miao G, Ostrowski RP, Mace J, Hough J, Hopper A, Peverini R, Chinnock R, Zhang J, Hathout E
2006 Dynamic production of hypoxia-inducible factor-1alpha in early transplanted islets. Am J
Transplant 6:2636-2643.

35) Xu X, Yi H, Kato M, Suzuki H, Kobayashi S, Takahashi H, Nakashima I 1997 Differential

sensitivities to hyperbaric oxygen of lymphocyte subpopulations of normal and autoimmune mice.
Immunology letters 59:79-84.

36) Song XY, Sun LN, Zheng NN, Zhang HP 2008 Effect of hyperbaric oxygen on acute graft-versus-
host disease after allogeneic bone marrow transplantation. Zhongguo shi yan xue ye xue za zhi /
Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of
Pathophysiology 16:623-626.
37) Jacobs P, Wood L, Van Niekerk GD 2000 Therapy: Hyperbaric Oxygen as the Only Effective
Treatment in Mutilating and Resistant Systemic Vasculitis. Hematology (Amsterdam, Netherlands)

38) MacKenzie DA, Sollinger HW, Hullett DA 2003 Decreased immunogenicity of human fetal
pancreas allografts following hyperbaric oxygen culture. Transplantation proceedings 35:1499-

39) Bowen KM, Andrus L, Lafferty KJ 1980 Successful allotransplantation of mouse pancreatic islets
to no immunosuppressed recipients. Diabetes 29 Suppl 1:98-104.

40) Sullivan FP, Ricordi C, Hauptfeld V, Lacy PE 1987 Effect of Low-Temperature Culture and Site of
Transplantation on Hamster Islet Xenograft Survival (Hamster to Mouse). Transplantation 44:465-

41) He YG, Niederkorn JY 1996 Depletion of donor-derived Langerhans cells promotes corneal
allograft survival. Cornea 15:82-89

42) Erdmann D, Roth AC, Hussmann J, Lyons SF, Mody NJ, Brown RE, Kucan JO 1996 Hyperbaric
oxygen and cyclosporine as a combined treatment regimen to prevent skin allograft rejection in
immunohistoincompatible mice. Annals of plastic surgery 36:304-308.

43) Estrada EJ, Valacchi F, Nicora E, Brieva S, Esteve C, Echevarria L, Froud T, Bernetti K, Cayetano
SM, Velazquez O, Alejandro R, Ricordi C 2008 Combined treatment of intrapancreatic autologous
bone marrow stem cells and hyperbaric oxygen in type 2 diabetes mellitus. Cell transplantation

44) ADA Statements. Standards of Medical Care in Diabetes—2007. Diabetes 40 Care 2007 30: S4-

45) Faleo G, Bocca N, Ricordi C, Molina J, Zahr E, Umland O, Molano RD, Pileggi A 2009 Prevention
of autoimmune diabetes onset in NOD mice by hyperbaric oxygen therapy. Diabetes 58:A327-

46) Nathan DM, Buse JB, Davidson MB, Heine RJ, Holman RR, Sherwin R, 43 Zinman B.
Management of hyperglycemia in type 2 diabetes: A consensus 44 algorithm for the initiation and
adjustment of therapy: a consensus statement from the American Diabetes Association and the
European Association for 1 the Study of Diabetes. Diabetes Care. 2006 Aug; 29(8):1963-72. No
abstract 2 available. Erratum in: Diabetes Care. 2006 Nov; 49(11):2816-8.

47) Mooradian AD, Bernbaum M, Albert SG. Narrative review: a rational 5 approach to starting
insulin therapy. Ann Intern Med. 2006 Jul 6 18;145(2):125-34. Review .

48) Irl B. Hirsch, Richard M. Bergenstal, Christopher G. Parkin, Eugene Wright, 9 Jr., and John B.
Buse. A Real-World Approach to Insulin Therapy in 10 Primary Care Practice. Clin. Diabetes 2005
23: 78-86.
49) 40. ADA Statements. Standards of Medical Care in Diabetes—2007. Diabetes 40 Care 2007 30:

50) Executive Summary of the Third Report of the National Cholesterol 13 Education Program
(NCEP) Expert Panel on Detection, Evaluation, and 14 Treatment of High Blood Cholesterol in
Adults (Adult Treatment Panel III). 15 Expert Panel on Detection, Evaluation, and Treatment of
High Blood 16 Cholesterol in Adults 17 JAMA. 2001;285:2486-2497.