REGENERATIVE MEDICINE WITH STEM CELLS FROM ADIPOSE TISSUE
(HADSCS) – HUMAN ADIPOSE STEM CELLS WITH ADIPOSE MICROFRACTURED CLUSTERS AND PRESERVED STROMA-VASCULAR FRACTION (SVF)
When we talk about the use of stem cells, we are often used to thinking that a stem cell must differentiate (i.e., transform another cell type) to replace something that has been lost (for example, in a wound). A logical consequence of this old paradigm is to think that there is almost a “pharmacological” logic, that is, the more stem cells I am going to put into a given tissue, the easier it will be to try to repair that tissue.
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What emerges to date are several “cold showers”: first, adult stem cells are multipotent (they don’t differentiate terminally, they don’t have any phenotypes that you would like to get in regenerative medicine, so no effective cardiomyocytes, no competent chondrocytes, no competent vessels, etc.). Secondly, when you insert millions of cells into a site of damage, very few are found in a short time (at least 5%, and the rest are dead); the old idea was based on the fact that somehow these cells, inserted into a suffering tissue, were going to die. This has further led to increased scientific research to expand these cells (make them multiply in an ex-vivo environment), which in effect become the subject of what regulators call an advanced medicinal therapy product.
In recent years we have realized that we are facing a very different reality: despite such negative conditions (few implanted cells that survive in the suffering tissue and few cells that do not differentiate), there is still a minimal and subtle repair effect (this is called the paracrine effect: the stem cell tends to repair the tissue because it sends instructive messages to the recipient tissue so this somehow triggers the self-healing process).
So if we want to enhance the healing process by the type of cells we want to transplant, what is the problem? To date, all the techniques that we possess to recover cells from a tissue are manipulation techniques in that they use enzymes to dissociate cells from any tissue, whether it is adipose tissue, bone marrow, or any other source, they use multiple and repeated centrifugations, and they use in vitro cooking; all of which destroys what is the micro-environment in which the stem cells live. In fact, stem cells do not “swim” in tissues, but are found in very specific structures, a micro-environment called the stromal vascular niche, which is a particular Nano topography. Only in that environment are cells able to understand the problem in a tissue and send appropriate messages to repair the tissue.
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ANTIBIOTIC EFFECT
In all outpatient and non-outpatient cases performed with the grafting of hADSCs and micronized adipose tissue, there has never been a case of infection. Researchers are currently investigating a possible antibiotic effect released by hADSCs: it would appear that stem cells from adipose tissue secrete an antimicrobial peptide called HCAP-18/LL37. The closest idea is to demonstrate that fat processing without centrifugation but with state-of-the-art biomedical devices can somehow mechanically activate these cells, i.e., the physical stimulus they undergo during washing and volume reduction leads to intracellular mechanisms of gene activation and subsequent release of certain molecules, including the peptide HCAP-18/LL37.
EMA – FDA – CURRENT LEGISLATIONS
On the regulatory aspect of the use of stem cells we are witnessing these two phenomena: EMA (European Medicines Agency) and FDA (Food and Drug Administration), which had considerable differences on the conception in stem cells and on the methods of their preservation, today present more or less the same standards, standards that are, however, constantly evolving, so much so that we no longer speak of GMP but of cGMP, to say that it is a constantly evolving regulation and that makes it substantially very difficult to be able to affirm something today and have the guarantee that the next month it is still so.
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In this new and innovative adipose tissue reduction device we are dealing not so much with stem cells but with a three-dimensional tissue that contains them; this is outside the regulations, that is, it is not an ATMP (Advanced Therapy Medicinal Products), that is, it does not require GMP. This is not the case for an expanded stem cell, i.e., cultured in vitro: in fact, it is considered subjected to major manipulation, and the cell-factory (tissue bank) must take care of expanding the cells, freezing them, and supplying them to the surgeon in the last step. Reducing adipocyte clusters while maintaining the three-dimensional structure is technically a transplantation of adipose tissue and not adult stem cells, which may well be done in the form of lipofilling, implemented since 1985 to the present.
The other major difference from the regulatory point of view is that since last year (year 2013) stem cells are not consolidable therapy products: it means compassionate use is prohibited, they cannot be used. Compassionate drug use means making a new, unapproved drug available to treat a seriously ill patient when no other treatments are available; Furthermore, compassionate use only pertains to a drug that has already been applied for a particular use that is being utilized for other uses.
But what I want to emphasize is that they are not drugs! Biological therapy is one thing, drug therapy is another. The very fact that they include the number of cells and the minimum effective dose in the validation criteria for a cell product is pure madness, these are people who have studied very little and would go back to their biology books and see this cold shower I was telling you about: that is, of so many cells inserted almost none remain, therefore it is not a problem of the dose-time, it is not a problem of how many cells I insert!
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With this kind of tissue strategy with micro-fractured fat clusters and preservation of the stromal vascular niche we are the cell-factory, the cells expand within our body and so it’s completely different precisely because they live in a tissue that is transplanted, so compassionate use is allowed, it can go to banks that don’t need the cGMP and autologous banking is allowed by law.
The traditional technique used to date, with regard to the processing of adipose tissue, involves centrifugation: this has two strongly limiting aspects; first, the mechanical force of centrifugation kills a good percentage of stem cells contained within the syringes; second, the separation and rejection of pro-inflammatory materials (oil) is severely limited, both because the mechanical force is only partially successful in separating the liquid, and because its subsequent removal is operator-manual, thus not standardizable.
The stem cell itself does not directly repair the tissue, but it is the trophic factors secreted by these cells that tend to act on the recipient tissue. So the classic question is, once these factors are identified, is it not possible to inject only these cytokines and then repair the tissue even in the absence of stem cell transplantation? The answer is no! Because surely the niche is a kind of “molecular biology laboratory” that is placed stably in a suffering tissue.
THE NICHE THEORY FOR FAT GRAFT SURVIVAL (TISSUE ENGINEERING)
STEM CELLS
The use of embryonic stem cells has created heated beats at the bioethical level, because it is clear that any experimentation on such cells closely concerns the origin of life: in fact, very often the extraction of stem cells from the embryo results in the destruction and death of the embryo itself; on the other hand, we find reproductive cloning, with all its safety reservations that have limited the application of therapeutic cloning pending greater clarity of information and totally reassuring scientific data. It should also not be forgotten that embryonic stem cells are being used to make genetically modified organisms, which can, however, lead to the understanding of some genetically based pathologies.
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Regenerative medicine, on the other hand, is based on the use of adult stem cells, so it is a safe medicine, it is a medicine that reopens the hope of a cure for those many pathologies to date destined to long drug treatments with limited efficacy and numerous side effects, it is a medicine that in the not so distant future may replace organ transplantation, it is a medicine that respects bioethical principles, it does not cause the death of any embryo, and it does not promote cloning.
Among adult-type stem cells, the ones that are most, known are those found in the bone marrow, in fact we have that of the hematopoietic line, whose potential has been exploited for years, for example in bone marrow transplantation in leukemias, in lymphomas.
The resounding discovery in recent years is that these cells are also present in adipose tissue, and they are present in large numbers; there are even more of them than in bone marrow percentage-wise. The difficulty is to extract them, that is, to get them out.
The extraordinary thing is that these cells, extracted and preserved with a particular device, have an enormous advantage: one, is that these cells are almost ubiquitous, that is, any one of us has a minimal amount of adipose tissue; two, it is quite easy to take them, just a localized and therefore totally minimally invasive mini-liposuction is enough to take a quantity certainly enough to do a cultivation of these cells, but also to have enough for immediate use.
They have very good biological characteristics, that is, they are cells that can still differentiate into all tissues, they can be banked for future use, so one takes them at 30 years of age and heals as he would heal at 30 years of age and not as he would heal at 70 years of age when he maybe gets a problem.
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These cells constitute an autologous tissue product, meaning its material that is taken and re-injected into the same patient, so it’s free of side effects, it doesn’t cause allergic reactions, and it’s not a painful technique because it’s administered through micro-punctures with a very thin needle-cannula.
These are cells that are very genetically stable, recent congresses such as the one in December 2012 in Rome and the second congress of the International Society of Plastic Regenerative Surgery (ISPRES) in June 2013 in Berlin, have shown that they are genetically stable even after several differentiations, and this is important for safety, which should always be the first concern of anyone involved in this field.
REGENERATIVE MEDICINE
The purpose of Regenerative Medicine and Surgery is not to replace what in the human body is no longer able to function, but to provide the necessary elements for its repair in vivo, to invent substitutes and principals that can merge with the human body and stimulate and support our body’s inherent ability to regenerate and heal itself.
Regenerative Medicine is an interdisciplinary field that has the perspective of repairing, replacing, and regenerating cells, tissues, and organs in order to restore certain anatomical, physiological, and biochemical functions that have deteriorated due to different causes (birth defects, diseases, trauma, and aging).
It has been estimated by the International Monetary Fund that the increase of only 3 years in the average life span of the world’s population will raise the cost of health services by 50 percent.
The ideal source of stem cells for clinical applications should ideally meet the following criteria:
- available in large quantities
- minimally invasive harvesting procedure
- harvested cells must be able to differentiate into different cell lines (e.g., stromal stem cells)
- cells must be able to be implanted safely and effectively, either as autologous or allogeneic
Adipose tissue is therefore considered the ideal source that meets these criteria.
Adipose tissue is one of the primary sources of mesenchymal stem cells (hMSCs). Regardless of the tissue of origin, they have demonstrated vasculogenic (vascular repair) fate in vitro and in vivo. They have the characteristic of being able to express a set of trophic factors (which contribute to nourishing the cell. Without them, the cell dies) secreted under different environmental conditions. These factors identify a paracrine peculiarity (they send messages from one cell to another in order to modify the physiology of surrounding cells), inducing angiogenic, antiapoptotic and antifibrotic responses.
ADSCs can be directed toward endothelial, smooth muscle, chondrocytic, hematopoietic, hepatocyte, and neuronal-like, osteoblastic, pancreatic, musculoskeletal, myocardial, and vascular fates.
1 g of adipose tissue contains approximately 5×103 MSCs, about 500 times greater than 1 g of bone marrow (bone marrow)
There are numerous examples in the literature demonstrating the in vitro multipotency of stem cells extracted from adipose tissue. Indeed, ADSCs are of mesodermal origin whose differentiation potential includes adipose, osteogenic, chondrogenic, and myogenic cells in addition to fibroblasts, tenocytes, and cardiomyocytes.
Other studies have also demonstrated the potential for differentiation into non-mesodermal lines, including neuron-like, endothelial cells, epithelial cells, hepatocytes, pancreatic and hematopoietic cells.