Frontiers in Stem Cell and Regenerative Medicine Research: Volume 4 E-Book
32,64 €
Mehr erfahren.
- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Serie: Frontiers in Stem Cell and Regenerative Medicine Research
- Sprache: Englisch
Stem cell and regenerative medicine research is a hot area of research which promises to change the face of medicine as it will be practiced in the years to come. Challenges in the 21st century to combat diseases such as cancer, Alzheimer and related diseases may well be addressed employing stem cell therapies and tissue regeneration. Frontiers in Stem Cell and Regenerative Medicine Research is essential reading for researchers seeking updates in stem cell therapeutics and regenerative medicine.
The fourth volume of this series features reviews on the use of stem cells through retrodifferentiation, mesodermal regeneration, hematopoiesis and mesenchymal stem cells. The volume also features a chapter on current knowledge on cell-based therapy in veterinary medicine.
Sie lesen das E-Book in den Legimi-Apps auf:
Seitenzahl: 498
Veröffentlichungsjahr: 2017
Ähnliche
Frontiers in Stem Cell and
Regenerative Medicine Research
(Volume 4)
Edited By
Atta-ur-Rahman, FRS
&
Shazia Anjum
BENTHAM SCIENCE PUBLISHERS LTD.
End User License Agreement (for non-institutional, personal use)
This is an agreement between you and Bentham Science Publishers Ltd. Please read this License Agreement carefully before using the ebook/echapter/ejournal (“Work”). Your use of the Work constitutes your agreement to the terms and conditions set forth in this License Agreement. If you do not agree to these terms and conditions then you should not use the Work.
Bentham Science Publishers agrees to grant you a non-exclusive, non-transferable limited license to use the Work subject to and in accordance with the following terms and conditions. This License Agreement is for non-library, personal use only. For a library / institutional / multi user license in respect of the Work, please contact: [email protected].
Usage Rules:
All rights reserved: The Work is the subject of copyright and Bentham Science Publishers either owns the Work (and the copyright in it) or is licensed to distribute the Work. You shall not copy, reproduce, modify, remove, delete, augment, add to, publish, transmit, sell, resell, create derivative works from, or in any way exploit the Work or make the Work available for others to do any of the same, in any form or by any means, in whole or in part, in each case without the prior written permission of Bentham Science Publishers, unless stated otherwise in this License Agreement.You may download a copy of the Work on one occasion to one personal computer (including tablet, laptop, desktop, or other such devices). You may make one back-up copy of the Work to avoid losing it. The following DRM (Digital Rights Management) policy may also be applicable to the Work at Bentham Science Publishers’ election, acting in its sole discretion:25 ‘copy’ commands can be executed every 7 days in respect of the Work. The text selected for copying cannot extend to more than a single page. Each time a text ‘copy’ command is executed, irrespective of whether the text selection is made from within one page or from separate pages, it will be considered as a separate / individual ‘copy’ command.25 pages only from the Work can be printed every 7 days.3. The unauthorised use or distribution of copyrighted or other proprietary content is illegal and could subject you to liability for substantial money damages. You will be liable for any damage resulting from your misuse of the Work or any violation of this License Agreement, including any infringement by you of copyrights or proprietary rights.
Disclaimer:
Bentham Science Publishers does not guarantee that the information in the Work is error-free, or warrant that it will meet your requirements or that access to the Work will be uninterrupted or error-free. The Work is provided "as is" without warranty of any kind, either express or implied or statutory, including, without limitation, implied warranties of merchantability and fitness for a particular purpose. The entire risk as to the results and performance of the Work is assumed by you. No responsibility is assumed by Bentham Science Publishers, its staff, editors and/or authors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products instruction, advertisements or ideas contained in the Work.
Limitation of Liability:
In no event will Bentham Science Publishers, its staff, editors and/or authors, be liable for any damages, including, without limitation, special, incidental and/or consequential damages and/or damages for lost data and/or profits arising out of (whether directly or indirectly) the use or inability to use the Work. The entire liability of Bentham Science Publishers shall be limited to the amount actually paid by you for the Work.
General:
Any dispute or claim arising out of or in connection with this License Agreement or the Work (including non-contractual disputes or claims) will be governed by and construed in accordance with the laws of the U.A.E. as applied in the Emirate of Dubai. Each party agrees that the courts of the Emirate of Dubai shall have exclusive jurisdiction to settle any dispute or claim arising out of or in connection with this License Agreement or the Work (including non-contractual disputes or claims).Your rights under this License Agreement will automatically terminate without notice and without the need for a court order if at any point you breach any terms of this License Agreement. In no event will any delay or failure by Bentham Science Publishers in enforcing your compliance with this License Agreement constitute a waiver of any of its rights.You acknowledge that you have read this License Agreement, and agree to be bound by its terms and conditions. To the extent that any other terms and conditions presented on any website of Bentham Science Publishers conflict with, or are inconsistent with, the terms and conditions set out in this License Agreement, you acknowledge that the terms and conditions set out in this License Agreement shall prevail.Bentham Science Publishers Ltd. Executive Suite Y - 2 PO Box 7917, Saif Zone Sharjah, U.A.E. Email: [email protected]
PREFACE
There continues to be tremendous progress in the field of stem cell and regenerative medicine research. The developments promise to change the face of medicine. This 4th volume of ‘Frontiers in Stem Cell and Regenerative Medicine Research’ should be of considerable interest to the readers as it presents state-of-the art reviews written by renowned experts in this fast moving field.
Ilham Saleh Abduljadayel presented a comprehensive review in chapter 1 on epimorphic regeneration and retrodifferentiation. Both processes have the capacity to recreate and reconstruct tissue with precise positional integration of cells in such a way that will enable healing without scars. In chapter 2 Fraser et al. extensively reviewed the unique genetic programmes that lead to mesendoderm formation and the pathways leading to mesoderm and endoderm specification. They also present examples where mature cell types from both germ layers interact to support their mutual development. These programmes are being employed to direct the differentiation of pluripotent cells in vitro into mesendoderm derived cells and tissues. Fraser et al. also reviewed the role of stem, progenitor and supportive cells within the hematopoietic tissues as essential elements of regenerative medicine in chapter 3.
Cell-based therapy is an emerging field in veterinary medicine that has been used for developing new therapies for degenerative diseases. In chapter 4 Izadyar et al. described different cell based therapies, their risks and benefits and their possible therapeutic use for veterinary medical applications. Mesenchymal stem cells (MSCs) are the most favored cellular candidates for regenerative therapeutics. Bhat et al. discussed how MSCs contribute to therapeutic efficiency include facilitating secretion of bioactive factors, induction of cellular recruitment and retention of progenitor faculties in the last chapter.
Knowledge of stem cell and regenerative medicine research continues to move ahead on many fronts. We hope that the readers will enjoy reading about the latest and stimulating development in this hot area in the 4th volume of this series.
We are pleased to place on record our heartfelt thanks to all the authors for their contributions. We are also grateful to the editorial staff of Bentham Science Publishers, particularly Dr. Faryal Sami, Mr. Shehzad Naqvi and Mr. Mahmood Alam for their constant support and great help.
Prof. Atta-ur-Rahman, FRS Honorary Life Fellow Kings College University of Cambridge Cambridge UK &Dr. Shazia Anjum Department of Chemistry Cholistan Institute of Desert Studies The Islamia University of Bahawalpur PakistanList of Contributors
Retrodifferentiation: From Concept to Bedside Stem Cell Therapy
Introduction
The ability to regenerate an entire complex tissue, organ or appendage upon damage is almost nonexistent in higher vertebrates, for instance in an adult human. This is because neither all tissues in the human body are endowed with stem cells that have the ability to proliferate and differentiate to replenish damaged or spent cells nor the adult human body possess sophisticated regenerative processes that enable replacement of body parts lost to severe injury. Most injuries are dealt with simply utilizing repair mechanism which entails closure of the injured site by deposition of fibrous tissue instead of cells. This leads to altering organ geometry and architecture including deterioration in function. In stark contrast, amphibians and particularly a selected group of urodele salamander can re-grow body parts, such as an amputated limb, even when old, in a process known as epimorphic regeneration. In this process, complex mechanisms, including dedifferentiation, innervation, positional integration and re-morphogenesis occur to restore the severed appendage. During the dedifferentiation phase, fully mature specialised cells from various positions around the circumference of the amputate, belonging to the mesenchymal lineage home to the stump site and dedifferentiate. This leads to the formation of a cluster of heterogeneous population of stem cells, known as the blastema. Homing and integration of mesenchymal cells occur according to positional values. This facilitates alignment and arrangement of cells of different lineages, in a configuration that upon re-morphogenesis permits restoration of the original geometry and architecture of the limb. Elucidation and understanding of epimorphic regeneration, including harnessing similar mechanisms in human, has tremendous applications in regenerative medicine than mere production of stem cells which at best proliferate and differentiate ex-vivo, but may fall short of positional integration or morph into tumors when transplanted into humans. The process which has been termed retrodifferentiation [1] is similar to the process of cellular dedifferentiation which facilitates blastema formation in salamander [2].
Retrodifferentiation of human leukocytes into a variety of pluripotent stem cell classes occurs in response to ligation of the monomorphic region of the major histocompatibility complex beta chain, using a monoclonal antibody (clone CR3/43) [3]. Each stem cell type generated is determined by the type of culture media and conditions utilized during retrodifferentiation. Similar to mesenchymal cell dedifferentiation in salamanders, leukocytes lose lineage associated markers, undergo homing and homocytic aggregation, and form heterogeneous stem cell colonies which subsequently redifferentiate into cellular components of the original tissue. Unlike salamander regeneration, retrodifferentiation is capable of transdifferentiation and histogenesis giving rise to entirely different tissues. In this process, mature mononuclear leukocytes can be converted into a variety of stem cell types belonging to the three germ layers: mesoderm, endoderm or ectoderm. Three hour human male retrodifferentiated haematopoietic stem cells (RHSC) have been shown to engraft in minimally irradiated NOD/SCID female mice [4]. Furthermore, 3 hr autologous RHSC were capable of long term engraftment in severe acquired aplastic anemia patients without any form of pre-conditioning therapy [5]. While in beta thalassemia major [6], a genetic blood disorder, the autologous RHSC were only able to ameliorate the course of the disease for six months. The ease by which any stem cell type can be prepared from human peripheral blood via retrodifferentiation, enabled the development of kits for the treatment of haematological and degenerative disorders, as a bed side stem cell therapy. In this manner, and in combination with leukopheresis and washing devices, the automation of stem cell production, will guarantee efficiency, sterility and specificity of stem cell infusate. Most importantly, retrodifferentiation recapitulates the process of histogenesis of various human tissues ex-vivo, thus paving the way to reconstructing organs by utilizing bio-printing devices in combination with various scaffold materials. Furthermore, the elucidation of the genetic events partaking in retrodifferentiation, transdifferentiation and histogenesis is paramount to regenerative medicine [7]. For example, the elaboration of cellular and extracellular pathways and signaling occurring during positional integration and histogenesis of a heterogeneous conglomerate of stem cells will enable us heal without scars, and mend tissue and organs with utmost precision and fidelity.
Healing Mechanisms in Response to Tissue Injury in Adult Human
Post cellular and tissue injuries, the human body mends or heals itself by utilizing two mechanisms: regeneration and repair. During regeneration, restitution of damaged cellular components of a given tissue, tend to be restored to normal configuration and function. On the other hand, the repair mechanism involves containment and closure of damaged tissue by deposition of collagen, which leads to scar formation with loss of normal architecture and deficit in function [8, 9].
Tissues capable of regeneration are those of the hematopoietic system [10] and epithelia of the skin and gastrointestinal tract. This is because they are endowed with stem cells capable of self-renewal and differentiation during injury or infection to replenish aged or damaged cells as long as the stem cell compartment is intact [11]. Tissues containing stem cells are known as labile tissues; for example, throughout the life of the haematopoietic system, there is a continuous renewal of this tissue, where injured, or aged specialized cells are replaced by stem cells, through proliferation and differentiation. In the process known as asymmetric cell division, the stem cells divide to generate daughter cells. One daughter cell embarks on differentiation to replace the damaged or spent specialized cell, while the other daughter cell, remains quiescent to prevent dwindling of the stem cell compartments in that tissue. The haematopoietic stem cells (HSCs) reside in the bone marrow of an adult human and consist of a heterogeneous population of stem cells with hierarchical development potential. For example, pluripotent/multipotent HSCs can replace granulocytes, megakaryocytes and erythrocytes, including platelets, through division and differentiation [12]. Other HSCs are either bipotent or monopotent, such as those giving rise to granulocytes / monocytes and erythrocytes only, respectively. As the HSCs divide and differentiate, apoptosis mechanisms operate to keep the population size of different lineage in check and in a specific proportion to each other [13, 14]. In other words, the haematopoietic system, though a proliferative tissue, renews at different rates during steady state and post injury, as long as the damage is not impacting various mechanisms associated with its proliferation development and apoptosis [15, 16].
The generation of various blood lineages, termed hematopoiesis, is regulated by the bone marrow microenvironment or niche. This niche regulates the quiescence, proliferation and differentiation of HSCs. The niche containing HSCs is widely distributed in the bone marrow and consists of perivascular and endosteal. The former consists of vascular endothelial cells, mesenchymal stem cells (MSCs), Cxcl12-abundant reticular (CAR) cells, perivascular stromal cells and Schwann cells, while the latter consists of osteoblasts. Niche factors include Stem cell factor (SCF; c-Kit ligand), thrombopoietin (Thpo or TPO) and Cxc112. Conditional deletion of SCF in endothelial cells and perivascular stromal cells results in a decrease in HSC number, suggesting an important role of these cells in HSC maintenance. In addition the physical environment, such as oxygen tension, temperature and contractile force also exerts a regulatory effect. During homeostasis, the majority of HSCs are relatively more quiescent than during infection stress, both proliferation and differentiation accelerate to neutralize and sequester pathogens [17].
Dysregulation and injury to the haematopoietic stem cells will either lead to over expansion or depletion of committed haematopoietic cell lineages, respectively. Cell loss has been observed in acquired aplastic anemia [18]; a hematological condition believed to be autoimmune in nature [19]. In this condition, bone marrow becomes gradually hollow or hypo-cellular, devoid of haematopoietic cells which are replaced by fat cells resulting in (i) low leukocyte count, (ii) deficiency in platelets, and (iii) red blood cell production leading to hemorrhage; and thus a propensity to contracting infection and, if left untreated, to morbidity. Alternatively, malignancy of the hematopoietic system results in clonal expansion of cells stuck at certain stages of development. Continuous proliferation of tumor cells leads to accumulation of genetic mutation(s) [20, 21], which transform the cells into aggressive clones capable of metastasis and obstruction of organs. In either scenario, cellular deficiency or over production, the underlying pathogenesis cannot be rectified spontaneously by the bone marrow and will require procedural intervention to replace defective cells [22].
Compensatory Regeneration
The ability of the liver to regenerate is recorded in the myths of ancient Greeks [23] despite there is no convincing evidence that they had any specific knowledge about liver regeneration. As recorded by the poet Hesiod (8th century BC), Titan Prometheus was subjected to the most severe punishment for his twice deception of Zeus for protecting the welfare of human kind. Firstly, he offered mortals the best meat from a slaughtered cow and gave the fat and bones to the gods. The second time, when an infuriated Zeus punished humans by taking fire, Prometheus stole it back for humankind. Prometheus was bound to the mountain Caucasus and subjected to perpetual torture by an eagle who fed from his liver each day, but the liver regenerated overnight. Peter Paul Rubens’ (1618) grand painting depicting a giant eagle piercing through Prometheus’ right hypochondrium and holding a portion of his liver in its beak is probably the most familiar illustration of the punishment of Prometheus used by lecturers on hepatic regeneration. Nearly 3000 years later, the work of Higgins and Anderson (1931) provided the first experimental evidence of liver regeneration following partial hepatectomy. Subsequently, many studies have shed light on how the liver regenerate at the cellular and molecular level.
While injuries to a stable or quiescent tissue such as the liver are met with compensatory regeneration, liver parenchyma exists in a non-dividing state and will enter the cell cycle in response to injury. For example, when a liver lobe is dissected for donation, the remaining liver cells will proliferate at such a rate that the liver reaches its original size [24] (before resection) albeit with similar cellular composition but without preservation of the original architecture or geometry of the liver master plan. While this kind of liver re-growth is a result of cell division, as in regeneration, but since it does not achieve return to the original form; it is called compensatory regeneration, although normal function is restored. Tissues falling into this category include parenchymal cells of the kidney, pancreas, lymphocytes, fibroblasts, endothelial cells and smooth muscle cells. Nonetheless, after persistent injury to the liver such as viral hepatitis, fibrosis is the end result of wound healing. This involves excessive accumulation of extracellular matrix proteins (ECM) including collagen, orchestrated by activated hepatic stellate cells, portal fibroblasts and bone marrow myofibroblasts. In advanced stages of fibrosis, the liver contains approximately six times more extracellular matrix (ECM) than normal, including collagens (I, III, and IV), fibronectin, undulin, elastin, laminin, hyaluronan, and proteoglycans [25]. The accumulation of ECM results from both increased synthesis and decreased degradation; and advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires a liver transplant [26].
In contrast, a nondividing tissue such as the myocardium is unable to regenerate; instead it is repaired by replacement of damaged cells by deposition of fibrous material, in order to reconnect the injured site. The adult heart has limited endogenous regenerative capacity [27] (stem cells), and in this instance, repair is achieved by patching the injured site with fibrous tissue [28]. This leads to a profound change in heart geometry and tensile strength. Prior to repair by connective tissue, an influx of inflammatory cells removes debris, leads to an exacerbation of inflammation and may augment matrix degradation resulting in cardiac rupture and weak scar formation, with reduced tensile strength and chamber dilation. Continued expression of pro-inflammatory mediators may impact non infarcted regions of the heart due to activation of pro-apoptotic pathways, enhancing fibrosis and deterioration in diastolic function [29].
On the other hand, adult wounds apart from minor cuts; injuries to fetal mammalian skin during early gestation result in non-scar wound healing. In humans, the window of non-scar wound healing terminates at approximately 24 weeks of gestation and is diminished at an earlier gestation age if wound size is large, as observed in fetal limbs [30]. In response to tissue injury, the fetal skin is capable of regenerating a seamless layer of collagen matrix that is identical to that of the original tissue [31]. In addition, dermal structures, such as sebaceous glands and hair follicles, form normally after fetal injury. The differences between fetal and adult skin wound healing appear to reflect the processes inherent to fetal tissue, such as the unique fetal fibroblasts, a more rapid and ordered deposition and turnover of tissue components, and particularly, a markedly reduced inflammatory infiltrate and cytokine profile.
Therefore, non-scar fetal wounds are relatively deficient in the inflammatory cytokine, Transforming Growth Factor β (TGF-β), including reduced expression of TGF-β1 and higher levels of hyaluronan in the extracellular matrix. In contrast, the fibrosis characteristic of adult wound repair may be associated with excess TGF-β. Experimental studies suggest that specific anti-TGF-β therapeutic strategies can ameliorate scar formation in adult wound repair and fibrotic diseases [32].
Epimorphic Regeneration
Humans are inept of more complex forms of regeneration, such as the replacement of an entire tissue, an amputated limb or dysfunctional organs; which are exact histological and morphological replica of the original. Urodele amphibians, such as axolotls salamanders and the eastern newt, Notophthalmus viridescens; have the unique ability to regenerate limbs, spinal cord, eye structures, and many vital organs through a process called regeneration. Two centuries ago, Spallanzani [33] was the first to describe regeneration in worms, snails, tadpoles and salamanders. The elucidation of his pioneering work is more relevant to regenerative medicine than the mere production of stem cells. The exact molecular mechanism behind epimorphic regeneration is not fully explored, though the cellular sequence of events involved in such a process are much clearer.
For example, in salamanders, after limb amputation and bleeding, the stump becomes covered by a fibrin clot within minutes of the injury. Subsequently, the blood clot becomes covered by migrating epidermal cells which originate by detachment from the basement membrane at the edge of the wound surface (Fig. 1). A layer of epithelium covers the wound within roughly 9 hours post injury [34]. This layer of cells, known as wound epithelium, (WE) [35] in subsequent regenerations becomes thickened to form the apical epithelial cap (AEC) [36]. Dedifferentiation ensues post epithelial wound healing, during which fully differentiated mesenchymal cells, including muscle cartilage and connective tissue proximal to the injury site; become undifferentiated by losing specialized characteristics [37 - 39]. Loss of differentiation is morphologically marked by rounding of nuclei, development of a prominent nucleoli, an increase in nuclear to cytoplasmic ratio and an increase in number of ribosomes [40]. The dedifferentiated cells [41 - 49], including resident stem cells, accumulate at the apex of the regenerate following proliferation to form mesenchymal cell aggregate known as the blastema. Blastema formation proceeds the establishment of the thickened AEC; a derivative of WE to facilitate the interaction of both cellular structures and to confer division signals to the blastema before embarking on redifferentiation [50 - 52]. In addition, before transition into AEC, the WE becomes innervated from the regenerating ends of an underlying nerve. Innervation induces the wound epithelium to become a specialized signaling centre that produces a number of growth or neuroptrophic factors and morphogens which act as chemoattractants and mitogens to attract and expand the blastema cell population. It is important to stress here that in the absence or obliteration of innervation, regeneration is stunted [53 - 57].
Furthermore, as regeneration progresses, the blastema undergoes morphogenesis and redifferentiation by transforming from a cluster of cells or small bud into elongated flattened palette-like structure. Redifferentiation occurs along a proximodistal axis from the amputation site, during which cartilage condensation and myogenesis occur first, followed by the differentiation of connective tissue, blood vessels and the nervous network [58]. Continued morphogenesis and growth of the regenerate give rise to a perfect functional replica, indistinguishable from the original limb; including the restitution of pigments which become fully formed shortly after. It is important to stress here that the initial patterning of the developing limb involves the sequential appearance of digits 1 and 2, followed by digits 3 and 4 [59]. During regeneration, all the digits seem to appear at once [60]. This rapid tissue regeneration is also observed during retrodifferentiation which will be discussed in more detail later on in the chapter.
Positional Integration and Molecular Mechanisms of Regeneration
Pattern formation of a limb or an organ, during development from an embryonic stem cell or during regeneration from blastema, occurs in an organized manner along a developmental axis during which cells cement with other cells in a specific spatial configuration [61]. This is because, cells possess positional identity of variable magnitude, across which they align themselves relative to each other to shape and form organs, appendages and various body parts in an organized three dimensional plan. This cellular property is epigenetically determined in human [62] and salamander [63] cells, and persists throughout adult life and is known as positional information. For example, grafting experiment performed by exchanging (host/recipient) blastemas from the same position between donor and recipient amputations on the limbs gives rise to an appendage at the same site [64]. However, exchanging blastemas from different positions on a limb will result in ectopic regeneration of an extra limb, not in the same location [65]. No graft grows by confronting a blastema from a completely different site on the body; such as head to limb exchange [66, 67], due to positional discontinuity and non-resolution through intercalary response.
Molecular insight into the mechanism of regeneration resembles the recapitulation of genes involved in limb development, examples are members of the Hox family [68], retinoic acid receptors (RARs) [69, 70], sonic hedgehog (shh) [71] and Tbox genes [72, 73]. Fibroblast growth factors and receptors are essential during wound healing and are secreted by the AEC and nerves, which upon denervation cause regeneration to fail, and this can be restored by exogenous application of FGF-2, FGF8 and FGF10 [74 - 80]. On the other hand, during the dedifferentiation phase, genes involved in extracellular degradation and remodeling appear essential, such as members of the matrix metalloproteinase (MMP) [81, 82] family; which may be key players in preventing scar formation and promoting destabilisation of the differentiated state. MMPs are essential for mammalian cell migration and proliferation, which may be important during migration and subsequent proliferation of mesenchymal cells, to form the blastema. Other extracellular proteases, such as thrombin and elastase [83 - 85], have also been implicated in dedifferentiation and extracellular remodeling. The homeobox gene, msx-1 [86, 87], has also been shown to have a key role in dedifferentiation. Surprisingly, ectopic expression of msx1, coupled with serum stimulation, can induce C2C12 [88] mouse myotubes to fragment and dedifferentiate in vitro into mononucleated cells which subsequently transdifferentiate into cells expressing chondrogenic, adipogenic, myogeneic and osteogeneic antigens.
Origin of Blastema
Genetic lineage tracing using a green fluorescent compound shows dedifferentiation of myofibers by fragmentation and conversion into proliferating Pax7 mononuclear cells [89], which integrates in the regenerating limb in one species of salamander Notophthalmus viridescens (newt). Whereas, in another specie, Ambystoma mexicanum (axolotl), myofibres neither generate proliferating cells, nor they contribute to newly regenerated muscle, instead resident PAX7+ cells (skeletal stem cells) provide the regeneration activity. Surprisingly, evidence of newt-like dedifferentiation was observed in specialized secretory cells of the epithelium of mouse trachea [90], which was demonstrated by ablation of CK5-expressing stem cells with doxyclycine, through inhalation and labeling of secretory cells expressing SCG1A with YFP. Lineage tracing of secretory cells, demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells expressing CK5. Single secretory cells clonally dedifferentiated into multipotent stem cells, when cultured ex vivo without basal stem cells. In contrast, direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation, albeit the propensity of committed cells to dedifferentiate was inversely correlated to their state of maturity.
Reprogramming the Differentiated State
Reprogramming differentiated cells into pluripotent stem cells can be achieved through nuclear transfer technology [91] or by forced expression of transcription factors inside specialized cells. During nuclear reprogramming, the nucleus of a differentiated cell is introduced into an enucleated oocyte, at a specific stage in the cell cycle, to re-jump start development to a totipotent stem cell state, capable of giving rise to an entire organism. Sir Gurdon was the first to demonstrate the plasticity of the differentiated state of gut epithelial cell nuclei which on transfer into enucleated eggs gave rise to adult frogs. His pioneering nuclear transfer experiment laid the basic background of cloning techniques, which started to gain interest after the birth of Dolly the sheep [92]. On the other hand, direct reprogramming of differentiated cells into an induced pluripotent stem cell (IPS) [93] state requires forced ectopic expression of genetic reprogramming factors. This is achieved by integration of genetic reprogramming material into differentiated cells: belonging to a single lineage; such as fibroblasts, using viruses, plasmids and modified RNA [94]. The difference between IPS and dedifferentiated blastema cells is that, reprogramming factors KLF4 (Kruppel-like factor) and C-myc (myelocytomatosis oncogene) are re-expressed in the latter, but without expression of embryonic stem cell antigens, as observed in the former [95]. In contrast to IPS produced from a single fibroblast, ablastema formed from a heterogeneous population of committed mesenchymal cells consists of a mixed population of stem cells, committed to various mesenchymal cell lineages. More similar to the process of blastema formation is retrodifferentiation. In this process, human leukocytes consisting of heterogeneous populations of mononuclear cells; when engaged with a monoclonal antibody against the homologous region of major histocompatibility complex (MHC) class II antigen beta chain revert to heterogeneous colonies of pluripotent stem cells, with hierarchical developmental potential. These retrodifferentiated stem cells can embark on two developmental routes, either redifferentiating into cellular components of the tissue of origin, or transdifferentiating into a new tissue altogether (Fig. 2).
Retrodifferentiation of Human Leukocytes
Retrodifferentiation is a process by which mature adult cells are converted into stem cells by cell surface receptor contact. This process is highly dynamic, during which differentiation or cell ontogeny is rewound backwards in a stepwise fashion in order for specialized cells to attain a stem cell state. Events involved in the gradual reversion from the differentiated state to stemness equate to rewinding a video clip backwards. For example, if development of a given stem cell involves acquiring sequentially characteristics A, B, C and D during forward differentiation, reversion to a stem cell stage involves losing characteristic D first, then C, then B before reacquiring characteristic A, which is a stem cell marker in this case (Fig. 3). This dynamic change in recapitulating development occurs rapidly, requiring sequential analysis of the reversion process, in order to account for every step in the conversion. Time lapse analysis of a retrodifferentiating heterogeneous population of cells belonging to different lineages and of known ontogonies are paramount to the elucidation of how differentiation is reversed to generate a heterogeneous population of stem cells. The most characterized tissue in the human body is the hematopoietic system, therefore deciphering retrodifferentiation in such complex tissue is much easier than others. Dedifferentiation (blastema formation) and retrodifferentiation are similar in many aspects. For example, specialized cell lose lineage-associated markers, migrate, and undergo homocytic aggregation, and once the heterogeneous stem cell cluster reach a certain size they embark on re-differentiation. However, retrodifferentiation is capable of transdifferentiation and histogenesis, giving rise to cell lineages not related to the original tissue. More importantly, retrodifferentiation occurs in vitro and in response to MHC class II beta chain ligation, which appears to emulate the stress equivalent to limb amputation in newts [96]. The difference between dedifferentiation and retrodifferentiation is most probably semantic, since sequence of events involved in the reversion process to a heterogeneous intermediary (Fig. 2) and stem cell states (blastema formation), is more difficult to elucidate in an in vivo complex situation. It should be stressed here that transdifferentiation of a mature adult cell refers to traversal of the differentiation barrier, adopting a completely different specialization fate. While redifferentiation refers to re-ontogony to the same specialization fate from where the reversion process started.
Phenotypes of the Retrodifferentiated Stem Cell State
Ligation of monomorphic regions of the MHC class II antigen on human leukocytes with monoclonal antibody CR3/43, under hematopoietic-inducing culture conditions, promotes the loss of more mature hematopoietic markers such as the pan leukocyte antigens CD45 or the B cell specific markers CD19. Extinguishing of these lineage-associated antigens occurs during cell migration and homocytic aggregation in a cell culture dish. These changes have been observed using confocal microscopy and immunostaining using anti-human leukocyte markers such as CD 19 FITC or CD45 FITC with CD34 PE-CY5 conjugated [97]. Within minutes of inducing retrodifferentiation, leukocytes gradually extinguish lineage-associated markers CD19 or CD45 while homing to form clusters of cell colonies. This is always accompanied by upregulation of the hematopoietic marker CD34 by migratory cells and during homocytic aggregation. Immunophenotypic analysis using flow cytometry of mononuclear cells (MNC) before and 2 hrs and 24 hrs after induction of hematopoietic retrodifferentiation shows a significant increase in the relative number of cells expressing the hematopoietic stem cell marker CD34. The majority of CD34+ cells are CD45 low and are either positive or negative for the CD38 antigen, typical of committed and more primitive haematopoietic progenitor cells, respectively [98]. The latter type has been shown to possess more long-term SCID repopulating potential. A significant proportion of CD34+ cells co-express c-Kit or CD133 which increases by 24 hrs as they enter the cell cycle. Furthermore, an increase in the absolute number of myeloid and erythroid progenitors occurs indicating resumption of redifferentiation. The retrodifferentiated CD34 hematopoietic stem cells can be purified using anti-human CD34 magnetic beads with significant CD34 expression. Seeding of retrodifferentiated hematopoietic stem cells in methylcellulose containing human recombinant growth factors [99] results in redifferentiation to give rise to a variety of hematopoietic colonies (Fig. 4) of different lineages such as colony-forming unit granulocyte, erythroid, monocyte, macrophage, megakaryocyte (CFU-GEMM), colony-forming unit granulocyte, monocyte, macrophage(CFU-GM), colony forming unit-monocyte, macrophage (CFU-M), blast forming unit-erythroid (BFU-E) and colony-forming unit erythroid (CFU-E). Interestingly, three hour retrodifferentiated hematopoietic stem cells derived from human male donors are capable of engraftment in minimally irradiated female non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Human cell engraftment ensues in the bone marrow, thymus and spleen. This animal model is considered the gold standard of demonstrating potency of human hematopoietic stem cells [100]. Further characterization of engrafted cells demonstrate the integration of significant levels of human lymphohematopoietic cell lineages, comprised of T-lymphocytes, monocytes, erythrocytes and megakaryocytes, including platelets. In addition, significant levels of clonogenic human CD34+ cells differentiation when seeded in methylcellulose containing human recombinant factors. The thymi of engrafted animals contained maturating human thymocytes, while the spleen mainly consisted of T-lymphocytes. Fluorescence in situ hybridization (FISH) further identified the presence of human male X and Y chromosomes at engrafted sites, whilst the human origin of the cells was confirmed by a specific PCR assay for the human Cart-1 gene.
Under neural/embryonic inductive culture conditions, and in response to ligation with CR3/43, reversion of leukocytes to colonies expressing OCT-4 and Nanog having flat morphology occur (Fig. 5) [101, 102] by 24hrs in culture, as analyzed using immunohistochemistry. Furthermore, analysis using immunostaining and flow cytometry showed significant increase in the number of undifferentiated cells [103] that are both CD45 and CD34 negative. These cells can be purified or enriched by negative selection on a Midi Macs column using anti-human CD45 and glycophorin A-coated magnetic beads to exclude human leukocytes and red blood cells. The unbound cells were negative for hematopoietic-associated markers that are typically expressed by the erythroid, lymphoid and myeloid cell lineages including MHC class I and II antigens when analyzed using flow cytometry and immunostaining. In addition, such purified cells transcribe high levels of the embryonic stem cell marker OCT-4, and, to a lesser extent, nestin and CD34 and stain positive with antihuman OCT-4, using immunohistochemistry. These cells formed or differentiated into embryoid bodies when cultured in hanging drops or seeded in growth factors-containing methylcellulose culture media (Fig. 5), respectively. Immunohistochemical analysis demonstrates expression of three germ layer markers: myocardial specific actin, alpha-fetoprotein, cytokertin 7 &20, desmin and s100.
Furthermore, induction of transdifferentiation on day 2 into neural cells occurs spontaneously on feeding with neural/embryonic inductive culture medium (without adding CR3/43 monoclonal antibody while feeding) (Fig. 5). This is marked by expression of nestin by cell colonies, accompanied by down regulation of embryonic stem cell markers OCT-4 and NANOG as determined by immunohistochemistry. Upon nestin expression, colonies start to lose their round shape and project neurites which continue to extend progressively with time and subsequently reconnect to each other. Upon maturation into neural axons, they connect with other axons emanating from nearby colonies or neurospheres. Dual immunohistochemical staining of cultures at defined time points using antibodies to MAP-2 (microtubule associated protein 2) and Tau (T proteins), NF (neurofilamnets) and GFAP (glial fibrillary acidic protein) and oligodendrocyte and CD45, analyzed by confocal microscopy, showed differential expression of neural, glial and oligodendrocytic antigens [104]. For example, by 48 hrs, cellular spheres were co-stained positive for MAP-2 and Tau, indicative of neurospheres containing immature neurons. One and 2 weeks later, the spheres differentiated into mature neurons, with MAP-2 being confined to cell bodies and Tau to the axons (Fig. 5). On the other hand, spheres that were analyzsed for NF and GFAP showed the presence of neural and glial precursors. However, 1 and 2 weeks later, such spheres differentiated into neurons expressing NF alone without GFAP, or astrocytic-like cells expressing GFAP without NF, the latter typical of glial cells. In contrast, co-staining for the pan-leukocyte marker CD45 and oligodendrocyte indicated the absence of CD45 expression at all-time points post retrodifferentiation, while oligodendrocyte staining was confined to the periphery of the spheres and persisted in 1 and 2 week old cultures. Moreover, time course analysis of neurotransmitters released by these neurons in response to depolarization revealed that, as they matured, the neurons produced increasing amounts of glutamate, GABA, tyrosine, dopamine and serotonin and uptake of taurine (unpublished data).
In contrast, 1 week post induction of human leukocyte retrodifferentiation and transdifferentiation under cardiopoietic [105] culture conditions grown on gelatinized six-well plates or chamber slides, cells transcribed the cardiac transcription factor GATA-4, human atrial natriuretic peptide (hANP) and cTnT. Dual immunohistochemical analysis of 1 week old cultures revealed the expression of myocardial-specific actin and cardiac-specific troponin I without CD45 expression. Three-day and 7-day human male leukocytes cultured under cardiac inductive conditions were capable of differentiating into fully mature cardiomyocytes when infused into non-irradiated Rnu/Rnu nude rats with needle stick damaged myocardia, and were integrated seamlessly into the area of damage. This was detected by XY chromosome in situ hybridization (FISH) and immunostaining for cardiac specific actin and HLA class I antigens (unpublished data).
Induction of human leukocyte retrodifferentiation under mesenchymal stem cell/ osteogeneic culture conditions (unpublished data) gave rise to colonies expressing the mesenchymal stem cell markers CD105, CD73 and CD90 after 24 hrs culture with a concomitant decrease in hematopoietic lineage specific markers CD45, CD3, CD 33 and CD19. After a week in culture (excluding CR3/43 antibody), cell colonies transdifferentiated into bone nodules, forming spheres which stained red with alizarin dye, and when immunostained with osteopontin and osteocalcin showed marked expression [106] (Fig. 4).
Induction of human leukocyte retrodifferentiation under endoderm-hepatogeneic culture conditions [107] gave rise to cell colonies expressing CD34 with CD45 after 24 hours in culture, which upon feeding transdifferentiated into hepatocytes (Fig. 6), expressing albumin and cytokeratin 18 after a week in culture (unpublished observation).
Clinical Application of Retrodifferentiated Stem Cells
Proofs of principle studies were carried out in two hematological conditions: acquired aplastic anemia and beta thalassemia major, following extensive analysis of the stability and safety of the retrodifferentiated hematopoietic stem cells (including karyotyping, g-banding and intradermal injection into mice). These studies showed no tumor development, and maintenance of normal karyotype even after 1 month in culture and 3 months in mice. Acquired Aplastic anaemia, is a life threatening rare bone marrow failure disorder. In this condition, depletion of the stem cell compartment, which is believed to be autoimmune in nature, gives rise to acellular bone marrow leading to hemorrhage, infection and severe anemia. The treatment of choice for this hematological condition is allogeneic bone marrow transplantation from fully HLA-matched sibling. Though this procedure is curative in the majority of young patients with aplastic anemia, extending this benefit to older patients or those lacking a family donor remains a major challenge. A proof of principle clinical study assessing the safety and efficacy of infusing autologous retrodifferentiated hematopoietic stem cells (RHSC) into four patients with aplastic anemia without the use of any pre- or post-conditioning regimen (including immunosuppression) was carried out. Without mobilization with growth factors, mononuclear cells were harvested from four patients by aphaeresis. These cells were induced to retrodifferentiate under hematopoietic culture conditions for 3 hrs. Autologous RHSC were washed and infused. Following single infusion, two patients became transfusion independent and have remained so for more than 10 years. Karyotyping and G-banding analysis pre and post-procedure in all patients remained the same. This pilot study was the first to demonstrate the functional utility of reprogrammed fully-differentiated adult cells in a clinical setting.
Beta-thalassemia [108] is a genetic red blood cell disorder affecting the beta-globin chain of the adult hemoglobin gene. This results in excess accumulation of unpaired alpha-chain gene products, leading to reduced red blood cell life span and the development of severe anemia. Current treatment of this disease involves regular blood transfusion and chelation therapy to lower blood transfusion–induced iron overload. Fetal haemoglobin (HbF) switching agents have been proposed to treat genetic blood disorders, such as sickle cell anaemia and betathalassemia, in an effort to compensate for the dysfunctional form of the beta-globin chain in adult haemoglobin (HbA). The rationale behind this approach is to pair the excess normal alpha-globin chain with the alternative fetal gamma-chain to promote red blood cell survival and ameliorate the anemia.
Infusion of autologous 3 hrs retrodifferentiated hematopoietic stem cells (RHSC) into 21 patients with beta-thalassemia was carried out. Follow up over 6 months showed a reduced mean blood transfusion requirement, increased mean fetal hemoglobin synthesis, and significantly lowered mean serum ferritin. This was always accompanied by an increase in mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). No adverse side effects in response to the infusion of autologous RHSC were noted. Since it was autologous, the procedure may have ameliorated the course of this genetic disorder through transient expression of HbF which was not maintained by 12 months post-treatment (unpublished data) due to reversion to adult hemoglobin synthesis. HbF to HbA switching occurs by 6 months in infants suffering from beta thalassemia [109].
More recently, a proof of principle clinical trial was conducted on patients with stage 4 liver cirrhosis having HCV infection. Though the results were preliminary, infusion of stem cells created under hepatogenic/endoderm culture conditions in these end stage patients significantly improved liver enzymes, such as albumin, ALT (alanine aminotransferase) and AST (aspartate aminotransferase), accompanied by increased % prothrombin concentration, alpha-fetoprotein and platelets counts. Up to six months post-treatment, fibroscan demonstrates amelioration of cirrhosis, changing from F4 to F3 in some patients. More importantly, the majority of patients did not require exogenous administration albumin due to non-occurrence of ascite (unpublished data).
Concluding Remarks
The ability of a group of specialized cells to reverse differentiate and give rise to blastema with the ability to redifferentiate and embark on remorphogenesis is tremendous. Equally significant is the ability of a heterogeneous population of human leukocytes to retrodifferentiate and form a heterogeneous population of stem cells with varied development potential. Moreover, pivotal to both mechanisms is the formation of cluster/s of cells capable of developing a precise three-dimensional configuration to offer the promise to reconstruct complex tissue or body parts. Retrodifferentiation enables the formation of human tissue ex-vivo, since it leads to transdifferentiation in response to altering culture media and conditions. Retrodifferentiation is a bona fide phenomenon, since rearranged immunoglobulin genes in B-cells and T-cell receptors revert to germ-line configurations in leukaemic cells and lymphoid cell lines [110]. This latter finding may be contentious, but evidence does exist. Preliminarily data involving sequential analysis of the transcriptional profiling of retrodifferentiated leukocytes transdifferentiated into neural tissue showed marked changes within 3 hrs of inducing retrodifferentiation. There were substantial changes in gene expression, up to 100-fold, associated with retrodifferentiation, which were visible as quickly as 3 hrs, with many sustained for 24 hrs. The transcripts changing during retrodifferentiation have previously been categorized, generically, in the ‘response to stress’ of the immune system, wounding, Wnt signaling pathway and pluripotency, senescence and autophagy, Matrix Metalloproteinases and pluripotency genes.
More specifically, pathways involved in integrin and focal adhesion, interferon signaling, interleukin signaling, and chemokines are the most strongly affected.
The conversion to the adherent state into nervous tissue is associated with more profound changes in gene expression, with at least 15 transcripts activated more than 100-fold. Conversion to the adherent state is associated with activation of extracellular matrix and matrix-binding proteins, and striking changes in chemokines, including expression of neural antigens and neurotransmitters including neurotransmission signaling pathways. Interestingly, transcripts such as matrix metalloproteinase (which is believed to prevent scar formation), immune response to stress and wounding, autophagy and senescence, and transcripts indicative of re-capitulating development have also been observed during epiomorphogenic regeneration.
More recently, emulation of blastema formation in aged human mesenchymal/ stromal cells by organizing cells in a 3-dimensional condensate [111] showed reversion of the differentiated state to an earlier stage of mesenchymal cell development as evidenced by switching from a CXCL12High CXCR4– to a CXCL12Low CXCR4+ Brachyury+ phenotype. An optimal autophagic response was required during dedifferentiation which promoted cytoplasmic remodeling, mitochondrial regression, and a bioenergetic shift from oxidative phosphorylation to anaerobic metabolism. Importantly, the dedifferentiated MSCs were capable of generating histologically-organized mesodermal structures when implanted in vivo; a property lacking in the parent, non-dedifferentiated MSC population grown in standard 2D conditions.
Finally, the ease by which human leukocytes can be harvested from peripheral blood using apheresis and the robustness of retrodifferentiation / transdifferentiation processes has allowed the design of single use kits for the production of a variety of stem cell types (manuscript in preparation). The automation of stem cells production for the regeneration of any tissue type with 100% efficiency, specificity and sterility will facilitate the wider application of stem cells therapy for many hematological and degenerative diseases for all age groups, thereby reducing waiting lists and the time to receiving stem cell therapy.
The ultimate goal of regenerative medicine is to regenerate without scars, reconstructing much needed tissue and complex organs to replace diseased or aged ones which can be achieved more readily and safely [112] by harnessing dedifferentiation or retrodifferentiation. For example, the combination of retrodifferentiation and transdifferentiation with bioprinting devices [113] will allow reconstruction of organs using a variety of scaffold materials which will propel regenerative medicine into human epi-morphogenesis with tremendous benefits and applications both ex vivo and in vivo.
CONFLICT OF INTEREST
The author confirms that the author has no conflict of interest to declare for this publication.
ACKNOWLEDGEMENTS
I would like to thank Dr. Keith Redpath for proof reading and editing of this chapter.
