EP2329011A1

EP2329011A1 - Use of isolated mature adipocytes in regenerative medicine for the reconstruction and reparation of tissues

Info

Publication number: EP2329011A1
Application number: EP09787797A
Authority: EP
Inventors: Saverio Cinti; Rita De Matteis; Riccardo Sarzani; Maria Cristina Zingaretti
Original assignee: Bint Srl Bio-Info-Nano-Tecnologie
Current assignee: Bint Srl Bio-Info-Nano-Tecnologie
Priority date: 2008-08-05
Filing date: 2009-07-21
Publication date: 2011-06-08
Also published as: WO2010016083A8; ITMI20081464A1; IT1394515B1; WO2010016083A1

Abstract

The present invention provides for the use of mature adipocytes isolated from the adipose tissue as multipotent cells in the regenerative medicine. The mature adipocytes isolated from the adipose tissue have been characterized before and after implant in the tissue of a recipient animal, showing a considerable plasticity in transdifferentiating into cell lines of a different nature than the original one as a response to suitable environmental stimuli.

Description

DESCRIPTION

"USE OF ISOLATED MATURE ADIPOCYTES IN REGENERATIVE MEDICINE FOR THE RECONSTRUCTION AND REPARATION OF

TISSUES" TECHNICAL FIELD OF THE INVENTION

The present invention finds application in the field of medicine and, in particular, in the regenerative medicine for the reconstruction or reparation of the tissues. STATE OF THE ART

In recent years, regenerative medicine has aroused great interest in the scientific community, and it is emerging as a new and revolutionary approach to the modern medicine . The regenerative medicine, in particular, is based on the use of viable cells for the restoring, maintenance, and enhancement of tissues and organs.

On the contrary, the practices of the "traditional" medicine, which is based essentially on the administration of drugs, hormones, enzymes, vaccines, or prosthesis implantation, such as artificial valves or plates, have several side-effects as well as limitations; for example, the prostheses in synthetic material may not integrate in the body,- the surgical reconstructions are often not much accepted, due to their invasive nature; the organ transplant requires the presence of a compati_ble donor, and it is not free from the rejection risk by the body of the recipient subject, finally, the immunosuppressor treatments are quite toxic and, therefore, not very much tolerated.

Instead, the regenerative medicine exploits the plastic properties and the regenerative properties of particular types of autologous cells, i.e. the body's own cells, that can be enhanced following the interaction with suitable extracellular matrices and growth and/or differentiation factors. In this manner, they can be exploited to stimulate the reparation of damaged tissues, restore their functions, or support the regenerative properties of the body, even if this is affected by disease.

The possibility that an already differentiated cell converts itself into another type of cell, as well as the mechanisms by which this occurs, have still not been completely elucidated, although in recent years scientific evidences have been accumulated, supporting the so-called plasticity of an adult tissue, as described, for example, by Wagers et al . in "Plasticity of adult stem cells", 2004 Cell, 116:639-648 or Slack JMV, in "Metaplasia and transdifferentiation: from pure biology to the clinic", 2007, 8:369-378. The achievement and successive maintenance by a cell of a particular development stage is determined by the combination of genes that it expresses, and it further depends on the combination of specific transcription factors, the expression of which is induced by the context in which the cell is present. Until a short time ago, instead, it was believed that all the cells deriving from the tissue development, such as mature cells, progenitor cells, tissue-specific stem cells, which are adult stem cells, would irreversibly keep the achieved specificity throughout the entire individual's lifetime. Therefore, according to this hypothesis, the progenitor cells and the adult stem cells would have the function of concurring to the tissue homeostasis by regenerating only those cells belonging to that specific tissue line, the differentiation into different cell lines not being possible .

However, recent studies have called this theory into question, demonstrating that the adult stem cells have the ability to "transdifferentiate" , generating cell progenies with lines that are different from those of the original tissue, as described also by Wagers et al . This property of the adult stem cells is due to their ability to respond in a different manner to different stimuli deriving from the micro-environment in which they are, and it has been by now demonstrated from a number of studies on different lines of adult stem cells. Some, in particular, would confirm that the stem cells that are present in the bone marrow can originate cells having a non-hematopoietic nature, such as myocardial cells, as described, for example, by Orlic D et al . in "Bone marrow cells regenerate infracted myocardium", 2001, Nature 410:701-705 or in skeletal muscle cells, as reported by Ferrari G et al . in "Muscle regeneration by bone marrow- derived myogenic progenitors", 1998, Science, 270:1528- 1530 or, again, in epithelial cells, as described by Krause DS, et al . , in "MuIti-organs, multi-lineage engraftment by a single bone marrow-derived stem cell" , 2001, Cell, 105:369-377. On the contrary, other works, such as Bjorson CR et al . in «Tumor brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo", 1999, Science 283: 534-537, report that cells of a non-hematopoietic nature, such as the stem cells that are present in the brain and muscle, could differentiate into cells of the hematopoietic line, while there are a number of studies describing the multipotentiality of the adult stem cells of mesenchymal nature, such as those isolated from the adipose tissue and which can give origin, if cultured under suitable conditions, to osteocytes, chondrocytes, myocytes, or neural cells, as reported by the studies of Zuk PA, et al . , "Human adipose tissue is a source of raultipotent stem cells", 2002, MoI Biol Cell, 13:4279-4295, and Fraser JK et al . , in "Fat tissue: an underappreciated source of stem cells for biotechnology" , 2006, Trends Biotech, 24 (4) : 150-154. Even if the collected data on transdifferentiation are not always reproducible, and a satisfying explanation to this phenomenon has still to be found, undoubtedly the finding of the versatility of the adult stem cells which, while having lost the totipotential nature that is typical of the embryonic stem cells, maintain the ability of differentiating into different cell lines, opened new possibilities for the regenerative medicine, allowing obviating to the many and cux-rently very pressing problems of a practical, medical, and ethical nature, associated to the use of embryonic stem cells.

Still preliminary studies suggest even the surprising possibility that also already-differentiated adult cells can transdifferentiate, thus losing, under physiological conditions, the markers and the typical functions of the progenitor line, to acquire the qualities of other cell lines. However, these phenomena have still to be thoroughly studied and explained. For example, it seems that the transdifferentiation is associated to the cell division only in some cases, as it has been found by Wagers AJ and IL Weissman, and it is to be clarified whether the phenomenon is associated to the cell de-differentiation or not, i.e. to that particular stage in which the cell does not have any peculiar characteristic which is typical of some cell lines. It shall be noted that the phenotypical change associated to the transdifferentiation is due to a different combination of active genes, this being to the detriment of another group of genes that are instead silenced, i.e. they are not expressed anymore. Therefore, if this modification of the pattern of active genes occurs slowly, the original phenotype converts into the phenotype of the transdifferentiated state, passing through the so-called de-differentiation stage. On the contrary, if the change of the expression of the gene pattern occurs more quickly, the cell does not take the de-differentiated stage; consequently, during a certain period of time, the coexistence of the expression of the genes that is typical of two different cell lines should occur: that of the cell before and after the transdifferentiation, respectively. For example, the direct transformation of the phenotype of differentiated cells belonging to adult animals is a phenomenon that has been generally ascribed to pathological conditions of an inflammatory and neoplastic type, or to other conditions that are specific of the embryonic development, as described by Eberhard D and Tosh D in "Transdifferentiation and metaplasma as a paradigm for understanding development and disease" 2008, Cell MoI. Life Sci. , 65, 33-40.

In the literature, important experimental criteria have been defined, on the base of which a transdifferentiation process can be defined; in particular, as proposed by Slack JMV and Tosh D, in "Transdifferentiation and metaplasia - switching cell types", 2001, Curr Opinion Gen Dev, 11:581-586:

- the two differentiated states, before and after the transdifferentiation, have to be clearly defined by a morphological and molecular characterization; and - a clear relationship between the two cell types by an in vitro experimental system has to be shown.

The adipose organ Offers a clear example of the plasticity attributed to the adult tissues. In fact, it has been extensively demonstrated by now that adult stem cells (Adipose Derived Stem Cells, hereinafter abbreviated as "ADSCs") are present in the adipose tissue, which cells are of a mesenchymal and multipotent nature, therefore capable of differentiating into different cell lines, such as adipocytes, chondrocytes, osteociti, neural cells, or myocytes, see Zuk PA, et al . , in "Human adipose tissue is a source of multipotent stem cells", 2002, MoI Biol Cell, 13:4279-4295, and Fraser JK et .al., in "Fat tissue: an underappreciated source of stem cells for biotechnology", 2006, Trends Biotech, 5. 24 (4) : 150-154. The concept of plasticity of the adipose tissue, besides following the identification of the ADSC cells, has been confirmed by the collection of a number of experimental evidences in favour of the ability of this tissue to respond to externals, nutritional, and0 environmental stimuli, with anatomical and functional changes .

Some studies , such as those of Murano I et al . , in "The adipose organ of SV129 mice contains a prevalence of brown adipocytes and shows plasticity after cold5 exposure", 2005, Adipocytes 1 (2) :121-130, have described particular transdifferentiation phenomena of the adult adipocytes in different cell types even in a reversible manner and under physiological conditions . These examples would represent a particular form of transdifferentiation0 that would involve a direct transformation of differentiated cells into cells of other cell lines, having a different morphology and function, therefore without passing through the de-differentiation stage. This phenomenon, which could be named as direct cell5 transformation, seems to be physiologic and reversible in the adipose organ. In fact, it has been hypothesized that the white adipose cells can transform into brown adipose cells_; a phenomenon that, from the physiological point of view, would involve a higher expenditure of energy and an improvement of the insulin sensitiveness. In small obese mammas, such as rats, this can be obtained pharmacologically, and it could aid to treat obesity and diabetes, as shown, for example, by Ghorbani et al in "Appearance of brown adipocytes in white adipose tissue during CL 316, 243-induced reversal of obesity and diabetes in Zucker fa/fa rats", 1997, Int J Obes Relat Metab Disord, 21 (6) :465-75. All this is made still more interesting by recent data, among which those published by- Needergard J et al . in "Unexpected evidence for active brown adipose tissue in adult humans", 2007, Am J Physiol Endocrinol Metab, 293 (2) :E444-52, identifying the brown adipose tissue in the adult human being.

Another interesting phenomenon of direct transformation in the adipose organ is the one occurring in the mammary gland during pregnancy and breastfeeding, as shown by the study of Morroni M et al . , in "Reversible transdifferentiation of secretory epithelial cells into adipocytes in the mammary gland" , Proc Natl Acad Sci USA, 101:16801 In the virgin female, this gland is 90% composed by adipose tissue, in which branched epithelial ducts are already present, originating from the nipple, while that part of the gland which is formed by lobular alveoles that are intended to the milk production and secretion develops during pregnancy. Therefore, also during breastfeeding there is a progressive reduction of the adipose component following the gland development, whereby, at the peak of the breastfeeding period, only few adipose cells are present, while more than 90% of the gland is represented by the epithelial lobuloalveolar structures and ducts. Instead, at the end of the breastfeeding, the adipocytes reappear, and in a short time the anatomy takes the features preceding the pregnancy, due to a progressive disappearance of the epithelial component. This remarkable example of tissue plasticity in the adult has been explained by some in the past by hypothesizing the loss of lipids in the adipocytes during pregnancy and breastfeeding, to which, at the end of the breastfeeding, a build-up step of lipids would follow. On the contrary, new studies have been produced in favour of the new theory about the reversible transformation of the adipocytes in milk- secreting epithelial cells, such as that of Morroni M et al. Furthermore, electronic microscopy and immunohistochemistry data, mainly based on the "fate mapping technique" (Cre-LoxP) , representing the technique of choice to prove the transdifferentiation phenomena, are. in favour of this theory; see, for example, Brian Sauer in Methods of Enzymology; 1993, Vol. 225, 890-900.

Therefore, such beneficial properties shown by the adipose cells could be advantageously used in the regenerative medicine, thus overcoming many of the limitations and problems that are associated to the techniques of the so-called traditional medicine. SUBJECT-MATTER OF THE INVENTION The devising of a method for the isolation and purification of adipocytes from the adipose tissue so as to keep the phenotype and plastic properties thereof unaltered represents an aspect of the present invention.

Furthermore, the present invention relates to the adipocytes as a medicament and in the cosmetic procedures; in particular, in the correction of defects of the soft tissues, of blemishes of the lips, or face, or breast .

DESCRIPTION OF THE FIGURES Fig. 1-A

Mammary gland ducts displayed by the "whole mount" technique and carmine staining. Figs. l-B-C-D

Histochemical analysis of samples of the mammary gland of an animal receiving the implant of adipose cells of the donor animal. The colorimetric reaction of the X- GaI test highlighted the presence of tissue deriving from the cells of the donor mouse in the middle of the native tissue of the recipient mouse's mammary gland. Figs. 1 E-F

Immunoreactivity of the recipient mouse's mammary glands (smaller boxes, right top) for casein and ADRPs, which are typical markers of the milk-secreting lobuloalveolar cells. DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to the method to obtain mature adipocytes from the adipose tissue .

Such method, in particular, allows keeping the native characteristics of the adipocytes unaltered, such as, for example, the pattern of expressed genes, showing itself through the expression of specific markers.

The experimental model that has been devised further allows analyzing the molecular events that are responsible for the mechanism underlying the transdifferentiation phenomenon.

More particularly, the adipose tissue explanted from transgenic mice has been implanted into wild type mice, i.e. mice not having any genetic modifications. To this aim, transgenic mice of the TgR(ROSA26) 26Sor strain have been employed (provided by Jackson Laboratory, Bar Harbor, Main, USA) . These animals express the la.eZ gene coding for the bacterial enzyme β- galactosidase in all the tissues of the developing embryo and in most of the adult tissues.

The enzyme β-galactosidase can be displayed through the X-GaI test . This test consists in a histochemical reaction allowing highlighting, by blue staining, the positive cells, i.e. bearing the la.cZ gene, and this allows controlling the fate of the donor's adipose cells through the assessment of the expression of such gene.

Samples of subcutaneous and visceral WAT have been collected from young male and female adult TgR(ROSA26) 26Sor mice. More precisely, the subcutaneous adipose tissue has been collected from 3 week-old male mice and from virgin female mice from the 4^th right mammary gland of the dorso-lumbar region, which was not infiltrated by epithelial ducts. By definition, the visceral adipose tissue is free from epithelial ducts; therefore, the absence of epithelial tissue in the subcutaneous adipose tissue samples has been verified by the "whole mount" technique, which allows displaying the ducts of the mammary gland formed by epithelial tissue by carmine staining (Fig 1-A) . Subsequently, the explanted tissue has been inserted via a syringe with 18 gauge needle (₁,024 mm diameter), under the capsule of the 4th mammary gland of singenic female mice at the first steps of pregnancy, and in virgin mice. At the end of the pregnancy, or in the first days after delivery, therefore during the breastfeeding period, the animals that received the explant have been sacrificed.

At least three experiments have been carried out for each of the following three experimental conditions:

- subcutaneous WAT obtained from the mammary gland of a female; subcutaneous WAT obtained from the mammary gland of a male; and

- visceral WAT of female animals.

Histochemical analyses of the collected samples have been performed to verify positiveness or not to the X-GaI test, as well as immunohistochemical analyses to verify the presence of markers that are typical of the epithelial cells of milk-secreting mammary glands: β- casein and ADRP (Adipose Differentiation Related Protein) .

Both positive and negative internal controls have been used, both of the native mammary gland and of the other organs of the recipient animal, in particular: the 4th contro-lateral mammary gland and the visceral WAT, heart, lung, liver, and abdominal skeletal muscle. Genotyping experiments have been carried out to sequencing the genome of the cells of the mammary glands receiving the explant and which resulted to be positive to the X-GaI test in order to confirm the presence or absence of the β-galactosidase gene. Such experiments have been carried out by using RT-PCR (Reverse Transcription-Polymerase Chain Reaction) , which is a technique largely known and widespread in the biology field. In order to characterize the nature and properties of the mature adipocytes, further experiments have been performed to confirm or not the sternness nature of these cells.

According to a further object of the present invention, the mature adipocytes described and obtained according to the method of the present invention find use in the procedures of regenerative medicine and tissue engineering and include, for example, the cell therapies devised to the aim of repairing and/or remodeling wounds and lesions of the soft tissues, such as lesions following surgical interventions, incidents, or burns; therapeutic procedures for the reconstruction of soft tissues, such as, for example, the reconstruction of the breast soft tissues after surgical interventions for the removal of tumoral masses, the reconstruction of adipose deposits and their metabolic functions in the cases of lipoatrophy due to congenital or acquired forms of lipodystrophy; cosmetic procedures for the correction of soft tissue defects, due, for example, to aging, or for the correction of blemishes of the lips, or face, or blemishes of the breast; cell therapies for the reparation of tissue damages of the muscular-skeletal system due to degenerative diseases, such as, for example osteoporosis or arthrosis, or due to trauma. The performed experiments have shown that the white adipose tissue, hereinafter abbreviated as WAT, of transgenic mice, suitably and specifically marked, if transplanted into singenic mice, i.e. provided with the same genetic background of the donor mice, during the pregnancy-breastfeeding stage, is capable of originating gland structures, the cells of which have the same marker that is present in the adipocytes of the donor tissue, while anyhow having the morphological and immunohistochemical characteristics that are typical of milk-secreting lobuloalveolar epithelial cells.

EXAMPLE 1

Purification of mature adipocytes

Subcutaneous adipose tissue has been collected by open biopsy (mouse and human being) or liposuction by using a cannula with a diameter of 3 mm and employing a pressure below - 500mmHg (human being) .

The collected tissue has been washed with HBSS (Hank's Balanced Salt Solution) and subjected to enzymatic digestion by incubation in HBSS and a solution of type II collagenase having a concentration of 2 mg/mL, at 37 ⁰C under stirring, for one and a half hours, by applying a concentration equal to 4 mg of enzyme solution per each gram of tissue. The portions of undigested tissue have been removed by filtration of the suspension obtained by the digestion through a Nylon filter having pores of 250 μτa. The mature adipocytes comprised in the floating fraction have been separated from the stroma- vascular fraction of the tissue by centrifugation of the cell suspension at 1000 rpm for 5 minutes. The floating fraction has been recovered, and the mature adipocytes have been resuspended in PBS (Phosphate Buffered Saline, i.e. a phosphate-buffered saline solution) . Then, the washing of the mature adipocytes was performed in PBS by centrifugation and recovery of the floating fraction. The washing procedure has been thus repeated thrice.

EXAMPLE 2

Analysis of mature adipocytes a) RT-PCR

The adipocytes obtained according to the procedure described in the Example 1 have been subjected to an analysis of the gene expression by means of the Reverse- Transcription Polymerase Chain Reaction (RT-PCR) technique. Besides the genes typically expressed in the mature adipocytes, among which the genes coding for leptin and adiponectin, it has been found that these cells express different markers that are typical of the stem cells, such as, for example, CD34, SCAl, CD90, and CD45.

In particular, these sternness markers resulted to be more highly expressed in the mature adipocytes than in the stroma-vascular fraction. b) Optical and electronic microscopy

The examination under optical and electronic microscope of the cells contained in the floating fraction of the stroma-vascular portion of the adipose tissue, separated after centrifugation, has excluded the presence of poorly differentiated cells; in fact, the cells had in an absolutely complete manner the morphology that is typical of the mature adipocytes, further showing, at an ultra-structural level, a good conservation of the organelles .

EXAMPLE 3

Transplant of the adipocytes

The adipocytes isolated and purified according to the procedure of the Example 1 have been inoculated by a syringe with 18 gauge needle (1,024 mm diameter) in the 4th mammary gland of "wild type" singenic virgin mice.

EXAMPLE 4 Analysis of transplanted adipocytes a) X-GaI test

During pregnancy or the initial step of breastfeeding, areas that are positive to the X-GaI test and being well circumscribed in the middle of zones of normally developed and X-GaI test-negative mammary gland tissue have been clearly identified in the recipient mice. The marked areas, therefore deriving from the donor mice cells, consisted in lobulo-alveolar glands surrounded by well differentiated adipose tissue, also marked (FIGS. 1 C and D) . b) Genotyp±ng

Genotyping experiments of the mammary glands receiving the explant confirmed the presence of the β-galactosidase gene . c) Immunohistochemical tests

Epithelial cells positive to the X-GaI test resulted immunoreactive for β-casein and ADRP, that are typical markers of the milk-secreting lobuloalveolar cells, while they resulted to be negative to the immunohistochemical marking with the MAC2 and F4/80 proteins, that are typical markers of the macrophages, and the a-smooth actin protein, which is a typical marker of the myoepithelial cells . d) Optical and electronic microscopy The observation under electronic microscopy of these samples revealed that these, lobulo-alveolar glands consisted in epithelial cells that had the organelles characteristic of the milk-secreting gland cells and that contained the typical electron-dense crystals, indicative of the reaction that occurred with β-galactosidase and identical to those observed in the lobulo-alveolar epithelial cells of the mammary glands of breastfeeding TgR(ROSA26) 26Sor females. e) Morphological examination The morphology of the lobulo-alveolar epithelial glands of the marked tissue appeared to be very similar to that of the surrounding native glands (Figs. 1 C and D) . f) Controls The native mammary gland and other organs of the recipient animal have been taken as positive or negative internal controls. In particular, as negative controls, the 4th contro-lateral mammary gland, visceral WAT tissue, heart, lung, liver, and abdominal skeletal muscle have been used. The blue glands, i.e. positive to the x-Gal test, appeared to be less differentiated that the native ones of the recipient mouse, due to the lower dimensions and lower immunoreactivity to casein and ADRP) (figs. IE and IF) .

Claims

₁. _A method for isolating and purifying the mature adipocytes from the adipose tissue of an animal, while keeping the phenotype and the plastic properties unaltered; said method comprising the steps of: a) Subjecting the sample to enzymatic digestion by using type II collagenase for a sufficient period of time; b) separating the stroma-vascular fraction from the floating fraction containing the adipocytes; and c) washing the adipocytes with a suitable buffer solution.

2. The isolated and purified mature adipocytes obtained according to the method of claim 1.

3. Mature adipocytes as a medicament.

4. The mature adipocytes according to claim 3 , wherein said mature adipocytes are obtained according to the method of claim 1.

5. Use of mature adipocytes in the cosmetic procedures .

6. The use of the mature adipocytes according to claim 5, wherein said cosmetic procedures include the correction of the defects of the soft tissues, of the blemishes of lips, or face, or breast.

7. The use of the mature adipocytes according to the claims 5 or 6, wherein said adipocytes are obtained according to the method of claim 1.