Clinical Trial Result: AdiStem Autologous Stem Cell Therapy for Patients with Idiopathic Pulmonary Fibrosis
Idiopathic Pulmonary Fibrosis (IPF) is a devastating, chronic lung disorder with complex and yet unknown disease biology for which there has been no treatment. A phase 1b, non-randomized, no placebo-controlled clinical trial using adipose derived adult stem cells was conducted on 14 patients between June, 2010 and September, 2011 at the Department of Pneumonology, Medical School, Democritus University of Thrace and University Hospital of Alexandropoulos, Greece with the approval of the Institutional Review Board and local Ethics Committee. Detailed safety monitoring indicated that cell treated patients – did not deteriorate in both functional parameters and quality of life indicators. Activation of the isolated stem cells was through autologous PRP and the use of Adistem’s Adilight low level laser irradiation. The result of this study has now been published in the Journal of Translational Medicine.
Click here to download the journal.
The FDA-approved method of 99mTC Radio Tagging was employed. AdiStem was able to clinically prove where Activated Stem Cells go once delivered back into the patient’s body. Below were scans taken for treatments done on particular cases where AdiStem technology was used.
Case Study 1: Brain Injury
When stem cells were injected via IV drip after activation using AdiStem technology, the activated stem cells went to the site of the injury. (See Figure 1.1.)
When stem cells were injected via IV drip without activation using AdiStem technology, the unactivated stem cells did not go to the site of the injury. (See Figure 1.2.)
Case Study 2: Dupuytren’s Contracture
The activated stem cells were administered via IV drip into the left arm. The stem cells went to the site of the arthritic right wrist. Reverse image show the same result. There were no visible stem cells in the left wrist.
Case Study 3: Lung Disease
When activated stem cells were administered via IV drip into the left arm, the stem cells went directly to the site of the inflammation in the lungs. Scan was taken 24 hours after stem cells were activated and radio tagged.
Case Study 4: Cerebral Palsy
Below is an Indium scan of a 9-year old male diagnosed with Cerebral Palsy. The patient’s own stem cells were harvested and activated. They were then tagged with Indium and returned the same day through an intravenous drip. A scan was taken and activated stem cells are clearly present in the brain on slide 15.
Below is a close up of slide 15. The yellow-orange color represents tagged stem cells that have homed on the area of the brain dysfunction.
PhotoActivation of Adult Stem Cells Derived from Adipose Tissue
Adult Stem Cells (ASCs), by definition, are unspecialized or undifferentiated cells that not only retain their ability to divide mitotically while still maintaining their undifferentiated state but also, given the right conditions, have the ability to differentiate into different types of cells including cells of different germ-origin – an ability referred to as transdifferentiation or plasticity.1,2 In vitro, the conditions under which transdifferentiation occurs can be brought about by modifying the culture medium in which the cells are cultured. In vivo, the same changes are seen when the ASCs are transplanted into a tissue environment different to their own tissue-of origin. Though the exact mechanism of this transdifferentiation of ASCs is still under debate, this ability of ASCs along with their ability to self-renew is of great interest in the field of Regenerative Medicine as a therapeutic tool in being able to regenerate and replace dying, damaged or diseased tissue.
Clinically, however, there are a few criteria that ASCs need to fulfill before they can be viewed as a viable option in Regenerative Medicine. These are as follows:3
Adipose Tissue Yields an Abundance of ASC’s
Compared to any other source, the high concentrations of regenerative cells found in adipose tissue (depots of fat for storing energy) especially in the abdominal region, by sheer volume of availability, ensure an abundance in number of ASCs ranging in the millions per unit volume. The sheer number available also has the added advantage of not needing to be cultured in a laboratory over days in order to get the desired number of ASCs to achieve what is called “therapeutic threshold” i.e. therapeutic benefit. In addition, harvesting ASCs from adipose tissue through simple, minimally invasive liposuction under local anesthesia is relatively easier and painless — and poses minimal risk to the patient compared to all other possible methods.
Adipose tissue ASCs (AT-ASCs) are extremely similar to stem cells isolated from bone marrow (BMSCs). The similarities in profile between the two types of ASCs range from morphology to growth to transcriptional and cell surface phenotypes.4,5 Their similarity extends also to their developmental behavior both in vitro and in vivo. This has led to suggestions that adipose-derived stem cells are in fact a mesenchymal stem cell fraction present within adipose tissue.6
Clinically, however, stromal vascular fraction-derived AT-ASCs have the advantage over their bone marrow-derived counterparts, because of their abundance in numbers – eliminating the need for culturing over days to obtain a therapeutically viable number – and the ease of the harvest procedure itself – being less painful than the harvest of bone marrow. This, in theory, means that an autologous transplant of adipose-derived ASCs will not only work in much the same way as the successes shown using marrow-derived mesenchymal stem cell transplant, but also be of minimal risk to the patient.
AT-ASCs, like BM-ASCs, are called Mesenchymal ASCs because they are both of mesodermal germ-origin. This means that AT-ASCs are able to differentiate into specialized cells of mesodermal origin such as adipocytes, fibroblasts, myocytes, osteocytes and chondrocytes.7,8,9 AT-ASCs are also able (given the right conditions of growth factors) to transdifferentiate into cells of germ-origin other than their own. Animal model and human studies have shown AT-ASCs to undergo cardiomyogenic 10, endothelial (vascular)11, pancreatic (endocrine) 12, neurogenic 13, and hepatic trans-differentiation14 , while also supporting haematopoesis15.
Low Risk to Patient
Autologous transplant of SVF AT-ASCs also poses extremely low risk to the patient when done as a single procedure in a sterile surgical operating room setting. Furthermore, it is postulated that SVF AT-ASCs due to their immunosuppressive properties may be transplanted into not only autologous but also allogenic tissues without initiating a cytotoxic T-cell response.16 We at AdiStem believe autologous transplant to be the safest and most viable option.
It is noteworthy that the protocol devised by AdiStem for the procurement of SVF AT-ASCs does not overlook the therapeutic potential conferred by the cocktail of ingredients present in the SVF. Let us look at this cocktail of cells, proteins and growth factors in a little more detail.
The extracellular matrix of adipose tissue contains different types of Collagen such as Types 1, 3-4, 7, 14-15, 18 and 27 to name a few.6 This is important in AdiStem’s Fat Transfer protocol where freshly isolated fat is used as a filler in augmentation or post-lumpectomy reconstruction of the breast and in the augmentation of the penis, and where collagen provides the structural support required for cell survival.
Furthermore, the extracellular matrix plays an important role in adipocyte endocrine secretions, and release of growth factors such as transforming growth factor beta (TGF-ß), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF), among others all of which are contained in the SVF.17 This is consistent with the secretions of cells in the presence of an extracellular matrix. The SVF also contains the various proteins present in the adipose tissue extracellular matrix of which Laminin is of interest due to its ability to help in neural regeneration.6
The cellular composition of the SVF ranges from pre-adipocytes to endothelial cells, smooth muscle cells, pericytes, fibroblasts, and AT-ASCs. Typically, the SVF also contains blood cells from the capillaries supplying the fat cells. These include erythrocytes, B and T cells, macrophages, monocytes, mast cells, natural killer (NK) cells, hematopoietic stem cells and endothelial progenitor cells, to name a few. The latter two types of cells, namely the hematopoietic stem cells and endothelial progenitor cells play important roles in supporting the viability of existing blood vessels and helping create new ones respectively.
We believe that these other ingredients that make up the SVF ‘cocktail’ act as an adjuvant to further augment the effect of the autologous transplant of SVF AT-ASCs.
Stem Cell Expansion is Unnecessary
An important point to note is that there is still debate whether freshly isolated ASCs are functionally similar to ASCs which have undergone expansion.18 We believe this debate to be of little consequence because of the high concentrations of regenerative cells we are able to harvest. Expansion is therefore unnecessary. Moreover, our own preliminary results in human subjects (n=32), where wound-healing was tested by the introduction of freshly isolated ASCs into the wound showed more than promising results. It must be stated however, that isolates from the lipoaspirate on its own proved less effective than when the isolates were introduced into either a proprietary Activation Medium containing known growth factor stimulators of stem cells in addition to the patients’ own platelet-derived growth factors (using PRP Kit) for one hour before being re-introduced into the patient.
ASCs Require Activation for Full Functionality
The observations stated above confirm that Adipose-derived ASCs though large in number lie dormant within the adipose tissue and that they require activation to come into full functionality for more successful implantation into the host tissue and to begin self-renewal by cell division and formation of other cell types by differentiation and transdifferentiation. This is also in line with the theory that ASCs are called into action only when the tissues within which they reside are dying, damaged or diseased. Further preliminary testing to increase the functionality of the Adipose-derived ASCs using specific frequencies of monochromatic light (LED Technology – AdiLight-2) – the specifics of which we prefer not to disclose at this time – has also revealed significant results.
AdiStem Phase I/II Clinical Trials in Humans on the Safety and Efficacy of Administration of Activated Autologous Adipose-Derived Stromal Vascular Fraction Adult Stem Cells are ongoing and at several stages of completion at various centers around the world for Management of Type II Diabetes, Breast Reconstruction Post-Lumpectomy, Management and Healing of Chronic Diabetic Ulcers and for Idiopathic Pulmonary Fibrosis.
Future research areas which have shown promising results in our initial case studies are Osteoarthritis, Emphysema, Stroke, Heart Failure and early stage Parkinson’s Disease.
Patents Have Been Filed
AdiStem Ltd. has filed multiple Australian Innovation Patents and multiple International PCT Patents on its methodology of extraction of adipose-derived ASCs from adipose tissue and various methodologies for activating ASCs.
Stem Cells and PRP
AdiStem Stem Cell Kits include standard PRP components. Growth factors (GFs) from the patient’s own circulating blood platelets are used to activate the adipose-derived ASCs harvested from the same patient.
Wound healing is a complex process, involving a mechanism of complex cascading regulatory events at both the molecular and cellular levels.19,20 Growth factors (GFs) are secreted by a wide variety of cells to regulate the wound healing process in an orderly manner.21,22 Over the last decade, various GFs, including platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-ß), have been used to accelerate the healing process.23-27
Platelet-rich plasma (PRP), as a storage vehicle of growth factors, is a new application of tissue engineering which was considered for the application of growth factors. PRP is a concentration of platelets in plasma developed by gradient density centrifugation.28 It contains many growth factors, such as PDGF, TGF-ß, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), etc.29,30 And it has been successfully used in a variety of clinical applications for improving hard and soft tissue healing.31-35 Platelet-rich plasma has also been shown to enhance the proliferation of human adipose-derived stem cells.36
The (stem cell) procedure involves the taking of blood during or just prior to the patient’s adipose tissue extraction procedure. Platelets are isolated from the blood and then activated to release their growth factors before photoactivation with AdiLight-2. The adipose-derived ASCs are then mixed with the growth factors containing plasma and activated in the AdiLight-2 for 20 minutes prior to being administered to the patient.
1. What are Stem Cells?
By virtue of their function, there are two distinct types of Stem Cells. One that is responsible for the development of an embryo from a single cell, by giving rise to specialized embryonic tissues, and resulting ultimately in the development of a human fetus; and the other which acts as the body’s repair mechanism by differentiating into specialized cells to replace damaged cells with healthy ones. These two types of stem cells are called Embryonic Stem Cells and Adult Stem Cells respectively. 1
Stem cells are different from other cells in the human body in two ways. First is that they are unspecialized or undifferentiated cells that have the ability to self-renew through mitotic cell division while still maintaining their undifferentiated state. This is called Symmetric cell division where both daughter cells retain the parent stem cell properties. 2
Second, is their ability to differentiate into different types of specialized cells while still maintaining their original numbers. Stem cells are able to do this through a process called Asymmetric Cell Division, where one of the two daughter cells differentiates into what is known as a progenitor cell, while the other daughter cell remains undifferentiated, thus retaining its parent stem cell properties. 2
Compared to a stem cell, a progenitor cell only has a limited ability for self-renewal; and following a limited number of rounds of cell division, the resulting progenitor cells differentiate into specialized cells of the body. 3
Research by Canadian scientists Ernest A. McCulloch and James E. Till in the 1960s saw the beginning of stem cell research. 2, 3
Fig.1: Stem Cell Division and Differentiation
A – stem cell
B – progenitor cell
C – differentiated cell
1 – symmetric stem cell division
2 – asymmetric stem cell division
3 – progenitor division
4 – terminal differentiation
Source: Wikepedia – Self-built
Permission: Public Domain
2. What are Embryonic Stem Cells?
Embryonic stem cells are cells which form an inner cell mass at the 4-5 day (Blastocyst) stage of human embryo development. They are responsible for the development of a human embryo into a human fetus by giving rise to specialized embryonic tissues. Embryonic stem cells are not present in the adult human and can only be obtained through the destruction of a human embryo.
3. What are Adult Stem Cells?
Adult stem cells (ASCs) are unspecialized or undifferentiated cells found in children and adult humans. These lie dormant (quiescent) and non-dividing within different adult human tissues until they are activated by signals from diseased, dying or damaged tissue to not only divide to form more stem cells, but also to differentiate into different types of specialized cells to replenish or regenerate these affected cells.
ASCs are generally ‘multipotent’ lineage-restricted cells with the ability to only differentiate into types of cells predetermined by the germ layer-origin of the tissue within which they reside. However, in vitro studies have shown that, given the right conditions, some ASCs can differentiate into cell types of germ-origin different to their tissue of origin. This is called Trans-differentiation or Plasticity. 4 , 5 , This makes these ASCs ‘pluripotent’ and hence very attractive in on-going stem cell research to find ways of culturing and transplanting healthy cells to replace diseased, damaged or dying tissues. 6
ASCs can be described in a number of ways depending on their potency, germ layer of origin, or their tissue of origin. For example, ASCs present in adipose tissue may be called Multipotent, Mesenchymal, Adipose-derived, ASCs. However, a more accurate description of ASCs harvested, isolated and activated using the AdiStem protocol would be to refer to them as Stromal Vascular Fraction-derived Adipose Tissue Mesenchymal Stem Cells (SVF-derived AT-MSCs)
ASCs are sometimes also referred to as Somatic (from Greek S?µat??ó?, of the body) stem cells.
4. What is Stromal Vascular Fraction?
Stromal Vascular Fraction (SVF) is the lipoaspirate obtained from tumescent liposuction minus the fat cells (adipocytes). Apart from adipocytes, the SVF contains a variety of other cells such as pre-adipocytes, endothelial cells, smooth muscle cells, pericytes, fibroblasts, and adult stem cells (ASCs). In addition, the SVF also contains blood cells from the capillaries supplying the fat cells.
These include erythrocytes or red blood cells, B and T cells, macrophages, monocytes, mast cells, natural killer (NK) cells, hematopoietic stem cells and endothelial progenitor cells, to name a few. Furthermore, SVF, in addition to the adipocyte endocrine secretions, also contains growth factors such as transforming growth factor beta (TGF-?), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF), among others. 7
This is consistent with the secretions of cells in the presence of an extracellular matrix. The SVF also contains the various proteins present in the adipose tissue extracellular matrix of which laminin is of interest due to its ability to help in neural regeneration. 8
5. Are there any issues with the use of Adult vs. Embryonic Stem Cells?
Embryonic stem cells can only be obtained by destroying a human embryo. Currently there are no clinical applications for embryonic stem cells. The use of these cells give rise to ethical and religious issues. However, there are no ethical issues with the use of ASCs because these cells can be obtained from adult human tissue; for example from liposuctioned adipose tissue (fat). Another important advantage of using ASCs is that these are autologous – one’s own cells – which the body will not reject. 9 ASCs from bone marrow have been successfully transplanted in sufferers of leukemia and related cancers for over 60 years now.
6. What are the sources of ASCs from one's own body?
Adult stem cells are present in all tissues in the human body. The major sources of ASCs that can be obtained from an adult human are:
7. Which is the best source to harvest ASCs from the human body?
The best source to harvest ASCs from one’s body is the adipose tissue. Below is a comparison of three sources:
8. Why is Adipose tissue the best source for harvesting ASCs?
The sheer number of ASCs that can be harvested at any one time from fat makes this the best source of ASCs in the human body. This number of ASCs harvested from fat also has the added advantage of not needing to be cultured in a laboratory over days in order to get the desired number of ASCs to achieve what is called “therapeutic threshold” i.e. therapeutic benefit. In addition, harvesting ASCs from adipose tissue is relatively easier, painless and poses minimal risk to the patient.
9.What is Autologous Adipose-derived Adult Stem Cell Transplant?
This is a four hour outpatient procedure 10 which involves the following:
10. How does AdiLight-2 work?
AdiStem Ltd. has researched the effect of different monochromatic light intensities and frequencies in the colored spectrum on various human and animal cell populations such as mesenchyme stem cells and white blood cells.
Low-level light photoactivation or photomodulation can be utilized for significant benefit in the stimulation of proliferation, differentiation, and inhibition/induction release of growth factors/cytokines of cells from any living organism.
The wavelength or bandwidth of wavelengths is one of the critical factors in selective photomodulation. Pulsed or continuous exposure, duration and frequency of pulses (and dark ‘off’ period) and energy are also factors as well as the presence, absence or deficiency of any or all cofactors, enzymes, catalysts, or other building blocks of the process being photomodulated.
Different parameters with the same wavelength may have very diverse and even opposite effects. When different parameters of photomodulation are performed simultaneously, different effects may be produced. When different parameters are used serially or sequentially, the effects are also different. The selection of wavelength photomodulation is critical as is the bandwidth selected as there may be a very narrow bandwidth for some applications — in essence these are biologically active spectral intervals.
AdiStem has ongoing international research projects looking at the effects of different frequencies of monochromatic lights on various cells including mesenchyme stem cells and white blood cells. It has now found five frequencies (three are present in AdiLight-2) that can activate stem cells, in vitro, and two frequencies that inhibit them. AdiStem has also found similar frequencies to modulate pro-inflammatory and anti-inflammatory cytokine release from peripheral blood white blood cells. AdiStem is also exploring the direct effect of different low-level frequencies of light on endogenous cells (in vivo).
AdiLight-2 is available from AdiStem for use in activating mesenchyme stem cells and modulating cytokine release by white blood cells.
Mesenchyme Stem Cells
When adipose-derived mesenchyme stem cells are taken out of a subject most of the cells are in a dormant state. In the body, stem cells and progenitor cells need to be activated by a physiological repair mechanism cascade, for example release of growth factor and chemokines by platelets. When the adipose-derived stem cells are photoactivated for 20 minutes with the AdiLight-2 device they show increased proliferation, increased production of integrins, vascular endothelial growth factor, thymosin beta 4 and interleukin 1 receptor antagonist. Hence, AdiLight-2 is of value in providing consistent clinical results, especially amongst age differences.
Peripheral Blood White Blood Cells
For many years internal medicine specialists in Eastern Europe and Korea have been using the photoactivation of blood, in vitro and in vivo, with various frequencies of light for immunomodulation in patients. When peripheral blood white blood cells (WBC) are photoactivated under AdiLight-2 for 10 minutes, an inhibition of pro-inflammatory cytokines (IL1, IL2, IL6 and TNFalpha) and induction of anti-inflammatory cytokines (IL1Ra and IL10) and beta endorphins are observed.
Reduces Pain and Accelerates Healing
Because of this property we have found AdiLight-2 to be a beneficial add-on to commonly used platelet rich plasma procedures in orthopedic and sports medicine procedures. One of the largest clinical drawbacks of the use of PRP in musculoskeletal healing is the aggravation of pain observed in the injected area post injection. Working with a group of Australian sports medicine specialists, we have deduced that a 10-minute exposure of WBC and platelets to AdiLight-2 prior to injection eliminates the aggravation of pain and potentiates the accelerated healing of PRP. It combines the benefit of autologous conditioned serum (ACS) with PRP in a simple 10-minute exercise.
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