Whitepapers

Greg Maguire, Peter Friedman

The SRM Molecular Foundry; BioRegenerative Sciences, Inc.

Corresponding author: gmaguire@srmfoundry.org

Abstract

The degree to, and the mechanisms through, which stem cells are able to build, maintain, and heal the body have only recently begun to be understood. Much of the stem cell’s power resides in the release of a multitude of molecules, called stem cell released molecules (SRM). A fundamentally new type of therapeutic, namely “systems therapeutic,” can be realized by reverse engineering the mechanisms of the SRM processes. Recent data demonstrates that the composition of the SRM is different for each type of stem cell, as well as for different states of each cell type. Although systems biology has been successfully used to analyze multiple pathways, the approach is often used to develop a small molecule interacting at only one pathway in the system. A new model is emerging in biology where systems biology is used to develop a new technology acting at multiple pathways called “systems therapeutics.” A natural set of healing pathways in the human that uses SRM is instructive and of practical use in developing systems therapeutics. Endogenous SRM processes in the human body use a combination of SRM from two or more stem cell types, designated as S2RM, doing so under various state dependent conditions for each cell type. Here we describe our approach in using state-dependent SRM from two or more stem cell types, S2RM technology, to develop a new class of therapeutics called “systems therapeutics.”

Introduction

The initial few days following fertilization of the human egg, all stem cells in the developing egg are able to create any tissue in the human body. However, about four days following the fertilization of a human egg, the stem cells in the blastocyst begin to differentiate and become pluripotent instead of totipotent (Cauffman et al, 2009). At this point in time, many different stem cell types are beginning to form that will have unique function related to the development, maintenance, and healing of various tissues throughout the body. The degree to which stem cells differentiate into specific adult phenotypes is only recently beginning to be understood. For example, cell types, such as the progenitor cell preadipocyte and adipose-derived mesenchymal stem cells, each of which was previously classified as one cell type, have now been shown to have phenotypic differences depending on the location of the preadipocyte niches or mesenchymal stem cell niches (Macotela et al, 2012; Ong et al, 2014). The signaling factors controlling the development and function of the stem cell types, and indeed the signaling factors that each stem cell type releases, are relatively unknown, but progress is being made. For example, we know that adult stem cells release hundreds of types of proteins (Sze et al, 2007) within the molecular pool, called the stem cell released molecules (SRM), and that each cell type will release a unique pool of molecules (Ribeiro et al, 2012; Baradis et al, 2014). These molecules in the SRM will prove to be important for developing many types of therapeutics, including, for example, immunoregulators for organ transplantation (Kato et al, 2014).

The more differentiated the stem cell, the more specialized the SRM will become. Further, that unique pool of molecules from one stem cell type can change in composition, including the types of molecular species, depending on intrinsic and extrinsic regulatory factors. For example, intrinsic factors related to simple passage number of a stem cell will change the composition of the SRM (Crisostomo et al, 2006; Deschne et al, 2014) and mesenchymal stem cells (MSCs) in different parts of the body will secrete unique pools of SRM (Park et al, 2009). Telomere dysfunction, whether the cause is intrinsic or extrinsic, will change the nature of the SRM (Ju et al, 2007). Likewise, preconditioning of human adipose-tissue-derived MSCs with the signaling molecule TNF-α had a profound impact on the SRM, and led to increased release of factors such as IL-6, IL-8, MCP-1, MMPs, PTX3, and Cathepsin L. (Lee et al., 2010). Further, signaling conditions during the immune modulating responses of human MSCs through Toll-like receptors (TLRs) on the MSCs leads to two basic phenotypic changes of the cells (MSC1 and MSC2) and a consequent dramatic difference in their SRM (Waterman et al, 2010).

Culture conditions can also have dramatic effects on SRM. A significant increase in the secretion of several arteriogenic cytokines, including VEGF, bFGF, PlGF, and TGF-β, was observed after subjecting hMSCs to 72 hr hypoxia compared with normoxic conditions (Kinnaird et al., 2004), while serum deprivation, another in vitro model for ischemia, has also been shown to induce secretion of angiogenic factors by hMSCs, although the results could have been attributed to in full, or part, to differences in cell proliferation rates (Oskowitz et al., 2011). Glucose levels have been shown to differentially affect the phenotype of endothelial progenitor cells and mesenchymal progenitor cells (Keats and Khan, 2012). Indeed, subtle variations in cell culture conditions can have significant consequences to the phenotype of stem cells (Bear, 2014).

The state of the extracellular matrix in the stem cell niche is also an important regulator of stem cell phenotype, where, for example, the absence of the SPARC protein in the ECM can drive hematopoetic stem cells into a state of quiescence (Ehninger et al, 2014). In the presence of antioxidants and FGF-2, adipose derived MSCs were shown to rapidly proliferate and retain their stem cell properties, and their osteogenic and adipogenic potentials were enhanced (Sun et al, 2013). Interestingly, new studies suggest that adult stem cells, and even somatic cells, may exist in a state of dynamic transition between different levels of potency (Tata et al, 2013; Kusaba et al, 2014) that is dependent on many factors, including paracrine and autocrine factors in the SRM from surrounding cells in the stem cell niche, and by the physical state of the stem cell niche (Su et al, 2013). The state of the oxidative stress in the stem cell may be a contributing factor in phenotype, including the state of pluripotency where the antioxidants curcumin and sesamin were shown to decrease oxidative stress and increase pluripotency (Yanes et al, 2010).

Stem cell derived control factors for determining the fate of stem cells and the potency of cells, including the dedifferentiation of somatic cells, their proliferation, and subsequent differentiation, may include GDF11, a protein secreted by bone marrow mesenchymal stem cells (Lai et al, 2010) that has been shown to be involved in stem cell fate and proliferation (Gokoffski et al, 2011), and has recently been shown to induce a number of regenerative effects, including neurogenesis (Katsimpardi et al, 2014). Recent studies also demonstrate that NANOG, a pluripotency transcription factor in embryonic stem cells, is also present in at least some adult tissues further raising the possibility that a dynamic state of pluripotency is a naturally occurring process in adults (Piazolla et al, 2014). Again, these shifts in the state of pluripotency will have concomitant shifts in the composition of the SRM released from the stem cell.

Given the differentiation of stem cells into distinct phenotypes, each of which releases a distinct pool of molecules with each distinct pool of molecules inducing a specific set of functions, a knowledge base of the secreted factors (SRM) from each stem cell type and the resulting actions from each pool of molecules will be instructive in the development of therapeutics. The resulting therapeutics that can be developed using the combination of many types of molecules has been termed “systems therapeutics” (Maguire, 2014). The “systems therapeutic” approach, where multiple molecule types target multiple pathways, is in contradistinction to the more traditional approach of small molecule development for perturbation of one pathway.

Moreover, as the stem cell types are cultured ex vivo in the laboratory and driven to state-dependent specific phenotypes through genetic, epigenetic, and other state-dependent variables, the concentration and composition of the SRM as a result can be experimentally manipulated for the purposes of therapeutic development. In addition, distinct pools of SRM from two or more stem cell types cultured in state-dependent conditions can be combined into a collective pool of molecules called S2RM, mimicking the collective actions of multiple stem cell types in their native state in the human body.

Two or More Types of Stem Cell Induce Healing

Cell replacement and regeneration occur in two basic forms: 1. renewal of spent cells during tissue homeostasis (homeostatic growth), and 2. responding to external injury, wounding, or amputation (epimorphic regeneration). A given healing process will require many actions acting through a well-orchestrated concert of mechanisms and molecules in the given tissue, and the result of this process depends on many factors, including the developmental age of the organism. Fundamental factors, such as caspases released from cells undergoing apoptosis, will activate both stem cells and progenitor cells in the wound healing process(Li et al, 2010), where apoptosis may be the driving force for cell proliferation during tissue regeneration a phenomenon termed “apoptosis-induced compensatory proliferation” (Fan and Bergman, 2008). Wounds occurring in early to mid-gestational fetal skin have been shown to heal through regeneration without the formation of a scar (Rowlatt, 1979), whereas adult wounds heal by a fibroproliferative response that emphasizes repair over regeneration. The complexity of this process, where fetal wounds differ from adult wounds in inflammatory responses, extracellular matrix (ECM) components, growth factor expression and responses, and profiles of gene expression is exemplified by the observation that adult skin in a fetal environment will still exhibit scar formation (Longaker et al, 1994). The state dependency of stem cells is so critical as shown in diabetes where the adipose stem cell niche in situ is altered, and the stem cells in the diabetic state are compromised in their ability to establish a vascular network both in vitro and in vivo (Rennert et al, 2014) where glucose itself has profound direct effects of stem cells (Cramer et al, 2010).

Wound healing begins at the moment of injury and involves both resident and migratory cell populations, extracellular matrix, and the action of soluble factors, including SRM. Stem cells and progenitor cells resident in the skin are certainly involved, but recruitment of stem cells from other sources, including bone marrow, is thought to be important (Harris et al, 2007; Tamai et al, 2011). The mechanisms underlying the processes described above involve: 1. inflammatory mediators and growth factors, 2. cell–cell and cell–extracellular matrix interactions that govern cell proliferation, migration and differentiation, 3. events involved with epithelialization, fibroplasia and angiogenesis, 4. wound contraction, and 5. remodeling. These mechanisms are initiated at the time of physical injury and proceed continuously throughout the repair process. Despite the processes of repair beginning immediately after an injury in all tissues, and that all wounds go through similar phases of healing, specialized tissues, for example, liver, skeletal tissue, and the eye have distinctive forms of regeneration and repair and follow separate pathways (Lawrence, 1998). Severe injury has been shown to increase the number of circulating stem cells (Fu and Liesveld, 2000; Kucia et al, 2004) and that these stem cells will participate in the wound healing process (Badiavas et al, 2003).

At the onset of trauma bone marrow stem cells will sense histamine released from platelets at H1 receptors and change their phenotype to one of releasing more IL-6 and more IL-8. The increased L-8 will attract polymorphonuclear neutrophil (PMN) cells, and the increased IL-6 will facilitate their survival through antiapoptotic functions (Nemeth et al, 2012). When the trauma inducing the injury has ceased, haemostasis has been realized, and an immune response set in place, the acute wound begins the tissue repair phase (Velnar et al, 2000). The proliferative phase starts on the third day after wounding and lasts for about 2 weeks thereafter. The process is characterized by fibroblast migration and deposition of newly synthesized extracellular matrix, building on the provisional network composed of fibrin and fibronectin. At the macroscopic level, this phase of wound healing can be clinically observed as an abundant formation of granulation tissue. The diverse processes that take place in the proliferative phase are briefly discussed below (Dieglemann and Evans, 2004).

Following injury, fibroblasts and myofibroblasts in the surrounding tissue are stimulated to proliferate for the first 3 days (Witte and Barbul, 1997). They then migrate into the wound, being attracted by factors such as TGF-β and PDGF, that are released by inflammatory cells and platelets (Goldman, 2004). Fibroblasts first appear in the wound on the third day after injury and their accumulation requires phenotypic modulation. Once in the wound, the fibroblasts proliferate profusely and produce the matrix proteins hyaluronan, fibronectin, proteoglycans, and type 1 and type 3 procollagen, all of which are released locally (Ramasastry, 2005) By the end of the first week, abundant extracellular matrix accumulates, which further supports cell migration and is essential for the repair process. Next, fibroblasts change to their myofibroblast phenotype. At this stage, they contain thick actin bundles below the plasma membrane and actively extend pseudopodia, attaching to fibronectin and collagen in the extracellular matrix. Wound contraction, which is an important event in the reparative process that helps to approximate the wound edges, then takes place as these cell extensions retract. Having accomplished this task, redundant fibroblasts are eliminated by apoptosis (Goldman, 2004).

MSCs are involved in all three phases of wound healing to varying degrees, whereby, for example, they recruit macrophages to the wound site (Chen et al, 2008), induce the fibroblast response to injury (Smith et al, 2010), and remodel the wound site (Hocking and Gibran, 2010), including a preferential release of collagen type III at the site (Fathke et al, 2004). While the MSCs contribute directly to wound repair by releasing molecules such as collagen to the wound (Fathke et al, 2004), the MSCs also act indirectly by releasing an instruction set to other cells thus initiating, for example, progenitor cell migration to the wound (Tasso et al, 2009). They also influence the wound’s ability to progress beyond the inflammatory phase and not regress to a chronic wound state. A significant component of the mechanism of action of MSCs is that they directly attenuate inflammatory response. Studies have shown that the addition of MSCs to an active immune response decreases secretion of the proinflammatory cytokines TNF-α and interferon-γ (IFN-γ) while simultaneously increasing the production of anti-inflammatory cytokines interleukin-10 (IL-10) and IL-4. It is these anti-inflammatory properties of MSCs that make them particularly beneficial to chronic wound treatment, as they can restart healing in stalled wounds by advancing the wound past a chronic inflammatory state into the next stage of healing. Accumulated data indicate the importance of MSC anti-inflammatory and immunomodulative activities in wound healing, detailed mechanisms of which are described in many reviews.

A number of studies have shown that MSCs have antimicrobial activity, critical for wound clearance from infection. MSC antimicrobial activity is mediated by two mechanisms: 1. direct, via secretion of antimicrobial factors such as LL-37 (Servold, 1991), and 2. indirect, via secretion of immune-modulating factors that will upregulate bacterial killing and phagocytosis by immune cells (Baum and Arpey, 2005). Further, the phenotype of macrophages can be regulated by MSCs into various M1 and M2 classes directed to either antimicrobial, phagocytic activity or one of controlling inflammation (Kim and Hematti, 2009).

MSCs in vivo can migrate to sites of injury in response to chemotactic signals modulating inflammation, repairing damaged tissue, and facilitating tissue regeneration. Furthermore, bone marrow stem cells home to the injury where cells in the wounded area secrete a protease that interacts with collagen matrix to produce a homing agent (Mauney et al, 2010). Differentiation and paracrine signaling are two key mechanisms by which MSCs improve tissue repair. MSC differentiation contributes by regenerating damaged tissue, whereas MSC paracrine signaling regulates the local cellular responses to injury. Current data suggest that the contribution of MSC differentiation of exogenous stem cells is limited due to poor engraftment and survival of MSCs at the site of injury, whereas the activation of endogenous stem cells may provide better results for the differentiation pathway (Arany et al, 2014). MSC paracrine signaling is likely the primary mechanism for the beneficial effects of MSCs on wounds, that is, to reduce inflammation, promote angiogenesis, and induce cell migration and proliferation (Greenhalgh,1998).

Analyses of MSC-conditioned medium indicate that MSCs secrete many known mediators of tissue repair including growth factors, cytokines, and chemokines, specifically VEGF, PDGF, bFGF, EGF, keratinocyte growth factor (KGF), and TGF-β. Stem cells are also known to release exosomes (Maguire et al, 2013), and exosomes from mesenchymal stem cells have been shown to contain factors, including miRNA, that switch cancer stem cells into a dormant state (Ono et al, 2014). Such a mechanism is important to dampen the cells in a wound from moving into a state of cancer (Bissell and Hines, 2011). Studies indicate that many cell types, including epithelial cells, endothelial cells, keratinocytes, and fibroblasts, are responsive to MSC paracrine signaling, which regulates a number of different cellular responses including cell survival, proliferation, migration, and gene expression.

MSC-conditioned medium acts as a chemoattractant for macrophages, endothelial cells, epidermal keratinocytes, and dermal fibroblasts in vitro. The presence of either MSCs or MSC-SRMhas been shown to promote dermal fibroblasts to accelerate wound closure. MSCs also secrete mitogens that stimulate proliferation of keratinocytes, dermal fibroblasts, and endothelial cells in vitro. Further investigation has shown that dermal fibroblasts secrete increased amounts of collagen type I and alter gene expression in response to either MSCs in coculture or MSC-conditioned medium . Overall, these data suggest that MSCs release soluble factors that stimulate proliferation and migration of the predominant cell types in the wound. In addition, the paracrine signaling of MSCs provides antiscarring properties through the secretion of VEGF and hepatocyte growth factor (HGF) and maintaining the proper balance between TGF-β1 and TGF-β3 . The molecular mechanisms of MSC involvement in wound healing are complex, and further details of these processes can be found in recent reviews. Stem cell niches in other regions of the body, including the hematopoietic stem cell niche, appear to be equally complicated as the skin stem cell niche with a rich interaction amongst many cell types, including a number of stem cell types and their respective SRM (Ehninger and Trumpp, 2011).

Naturally Induced Pluripotent Stem Cell (NiPSs) Within The State Dependent Stem Cell Niche
Natually occurring endogenous iPSs, or naturally induced pluripotent stem cells (NiPSs) occur within the state dependent stem cell niche. The concept of dedifferentiation seems to be an important adaptive mechanism in both the animal (Echeverri and Tanaka, 2002) and plant kingdoms (Grafi and Barak, 2014). In addition to the therapeutic development of embryonic stem cells and iPSs, the use of adult stem cells and the molecules that they release have been intensively investigated and have current therapeutic applications. Further, the molecules released from stem cells or neighboring cells, such as ciliary neurotrophic factor (CNTF), have been shown to transform myogenic lineage-committed myoblasts at a clonal level to dedifferentiate into multipotent progenitor cells that were then able to differentiate into several new phenotypes (Chen et al, 2005).

The endogenous mechanisms of adult stem cells, and possibly somatic cells in the stem cell niche, seem to include the ability to reprogram themselves into more primordial states that are pluripotent. That is, the adult stem cell, and even somatic cells, may exist in a state of dynamic transition between different levels of potency that is dependent on many factors, including paracrine and autocrine factors in the SRM from surrounding cells in the stem cell niche, and by the physical, chemical, and electrical state of the stem cell niche (Mammoto et al, 2013; Liu et al, 2013; Rhouabiha et al, 2013). Recently, treatment with reversine, a type of purine, transformed 3T3-L1 preadipocytes into MSC-like cells, as evidenced by the expression of MSCs marker genes. The transform allowed differentiation of lineage-committed 3T3-L1 preadipocytes to osteoblasts under the osteogenic condition in vitro (Park et al, 2014). Beyond transcription factors contained in the SRM, physical manipulation through the cytoskeleton is known to transmit signals to the chromatin and reprogram cells, and may represent an additional means for driving cells to varying levels of potency. Reprogramming of differentiated cells to stem-like cells has been described in several tissues and is well studied in the epithelial-mesenchymal transition where a differentiated epithelial cell transforms to a mesenchymal cell with a stem cell-like phenotype. Thus, by understanding adult stem cell function, we may develop the means to use these cells in many ways to maintain and heal the body, including a means of controlling naturally occurring iPSs.

The physical, chemical, and electrical state of the stem cell niche will have profound influences on stem cell function. Alterations of the stem cell niche in diseases such as diabetes will decrease the ability of endogenous stem cells, or autologous administered stem cells, to increase neovascularization and promote wound healing (Rennert et al, 2014).

In Figure 1, we see levels of interactions that may control the natural iPSC state. Considering wound healing as described in the aforementioned section, many factors, such as histamine, an important regulator of cell fate, including neurons (Bernadino et al, 2012; Panula et al, 2014), are released at the site of injury. As an example of the actions of these factors, histamine will activate TRPM4 calcium channels in the mesenchymal stem cells and bias the dynamic transition of the stem cells toward differentiation into the needed mature cells types at the injury site (Tran et al, 2014), including osteoclastogenesis (Biosse-Duplan, 2009). Similarly, exposure to sunlight will stimulate vitamin D3 levels and induce differentiation of stem cells, doing so through a downstream pathway that includes histamine (Pochampally , 2007).

Reprogramming of cells to push the dynamic transition towards more potency has been specifically shown in mammalian cells whereby muscle cells(Mu et al, 2011) and pancreas cells (Tellez and Mantaya, 2014) will dedifferentiate into a more pluripotent state following Injury, and where fibroblasts were incubated in cell extracts of adipose-derived stem cells. The fibroblasts displayed pluripotent gene expression that was associated with the loss of repressive histone modifications and increased global demethylation. The genes Col1a1 and Col1a2, which are typically found in differentiated cells only, demonstrated decreased expression and increased methylation in the 5′-flanking regulatory regions (Rong, 2014). Of the many factors released by mesenchymal stem cells, microRNA is one of the factors that have been shown to induce pluripotency in mouse and human somatic cells (Anoyke-Danso et al, 2011). In general, stress is a key factor that can naturally induce pluripotency. For example, simple isolation of mammalian cells from contact with other cells and their normal niche, originally exhibiting a limited differentiation potential, may become multipotent (Shoshani et al, 2014). Pluripotent cells can reside in the naïve state or the primed state where the naïve state is more potent than the primed state (Nichols and Smith, 2009). Dedifferentiation under hypoxic conditions can drive committed cells beyond the primed state fully back to the naive state of potency where the pluripotent cells are then capable of forming teratomas (Mathieu et al, 2013).

Cancer cells and pluripotent stem cells follow certain common rules. Both cell types, when placed in a dysregulated extracellular matrix, will exhibit an increased state of potency . Cancer cells, when returned to a regulated extracellular matrix (ECM), will revert to a normal phenotype (Bissell and Hines, 2011; Booth et al, 2011). Likewise, dedifferentiation of cells into a pluripotent state can occur when the cell is isolated and looses connections with other cells and the ECM (Shoshani et al, 2014), and stem cells that have differentiated can revert to a more pluripotent state by changes in the concentration of the ECM associated protein, L-proline (Comes et al, 2013). Thus, induction of pluripotent stem cells is a naturally occurring phenomenon that can be controlled in vivo for therapeutic effect by manipulating the state of the stem cell niche.

The Concept of a Systems Therapeutic

Diseases are not a simple consequence of abnormality in one pathway, or even at one level of the organism, such as at the level of genes. Rather, disease reflects the perturbations of the complex system of intracellular networks acted on by complex environmental regulators. Much of previous work to understand disease and drug response traits have focused on single dimensions of the system. Achieving a more comprehensive and predictive understanding requires examining living systems in multiple dimensions and at multiple scales. Although biological engineering principles are necessary, with the necessity to remove unnecessary complexity for the development of a particular therapeutic, the individual components of complex systems are so tightly coupled that the components cannot be analyzed in isolation. This predicament in biology, such as the desire to place the sequencing of the genome as the singular predictor of disease, is similar to that dictum in physics where electrodynamics was broken down into the misbegotten particles and fields theorem by Bohr and his Copenhagen interpretation(Mead, 2013). Biological complexity is an extreme example of complexity, and arises from the inclusion of active, plastic components, flexible design principles, nested feedback loops, component multi-functionality, and multiple layers of system dynamics developed through evolutionary processes that are, at least partially, driven through environmental regulators. The power of the dynamic biological system has been recognized in engineering where, for example, neuromorphic engineering (Mead, 1989) has become an important player in the development of new computer chip technologies such as TrueNorth (Merolla et al, 2014).

Even with the introduction of systems biology to the fields of biology and therapeutic development, the mindset in therapeutic development has often remained one of using systems biology for finding the one pathway, or the one target, that is best perturbed to develop the therapeutic. “Finding the magic bullet” is a common phrase that describes this common problem. Instead, the correct thinking needs to shift to one of finding the minimum set of pathways, or the minimum set of targets, using the “minimum molecule set” to perturb in order to best develop a therapeutic. That is, biology is a system, and a particular disease state is the result of multiple perturbations in that system, not just one perturbation. Therefore, only through a thorough understanding of biocircuits in normal and disease states, and using computationally intensive biological design-build-test-analyze cycle, with therapeutic molecule production batches based on this process, can we hope to develop safe and efficacious therapeutics through a multi-targeted, “systems therapeutic” approach. The approach then is to use a reductionist set (system) of molecules, the minimum molecule set (MMS), that is not overly reductionist so as to be ineffective, but instead use the least number of necessary molecules that are sufficient to realize a safe and efficacious therapeutic. The notion that human diseases are the result of complex interactions among networks has significant implications for drug discovery, leading to the design of molecule combinations that impact entire network states rather than designing drugs that target specific disease associated genes.

Development of Systems Antimicrobials

The attempt to develop animal-derived antimicrobials is not new. For example, in the 1990s great hope, and many dollars spent, was placed on the development of a small peptide from frog (Xenopus laevis) skin as an antibiotic (Zassloff, 1987). The observation that frog skin heals itself, despite the frog living in a very septic environment, led to the formation of Magainin Pharmaceuticals. After years, and millions of dollars, spent on development and Phase II clinical trials, today Magainin’s assets are the auction block (Magainin changed names to Genera and then liquidated: http://www.fiercebiotech.com/press-releases/genaera-corporation-announces-approval-plan-liquidation-and-dissolution-board-direc-0). Why? Because the frog’s skin does not heal itself through a reductionist approach with only one molecule (a peptide), and Magainin didn’t fully learn the frog’s lesson. The lesson not learned was that Magainin developed their antibiotic based on one peptide, a reductionist approach, instead of a mix of antimicrobial factors, a systems antimicrobial approach.

Lipids were first demonstrated by Koch (1881) to have antibiotic activity, and exists in human skin, for example, as a wide range of molecule types comprising a significant part of the innate immune system (Fischer et al, 2012; Thormar et al, 2013). Like Magainin, a similar reductionist approach was used in the development of squalamine, a lipid compound (aminosterol) derived from the dogfish shark (Squalus acanthias). Squalamine was initially discovered on the basis of its anti-bacterial activity, and has broad spectrum antimicrobial activity against fungi, protozoa, and many viruses (Moore et al, 1993). Sadly, isolated squalamine was never approved for antimicrobial use and is now sold as a nutritional product by a number of companies in capsule form. Once again, the “Copenhagen reductionist” approach to therapeutic development has failed us. Here again, instead, an approach to developing antimicrobials using a collection of molecules, including peptides and lipids, is in development.

Development of Cancer Systems Therapeutic

Cancer is strongly associated with a deregulated ECM (Lu, Weaver, Werb, 2012). While cancer and stem cells are regulated by many factors, both cancer cells and pluripotent stem cells follow certain common rules such as regulation by the ECM. Both cell types, when placed in a dysregulated extracellular matrix, will exhibit an increased state of potency . Cancer cells, when returned to a regulated extracellular matrix (ECM), will revert to a normal phenotype (Bissell and Hines, 2011; Booth et al, 2011). Likewise, dedifferentiation of cells into a pluripotent state can occur when the cell is isolated and looses connections with other cells and the ECM (Shoshani et al, 2014), and stem cells that have differentiated can revert to a more pluripotent state by changes in the concentration of the ECM associated protein, L-proline (Comes et al, 2013). Given that that the ECM can act through mechanical and biochemical mechanisms to regulate the cancer phenotype, one important means to revert the cancer phenotype to the normal somatic cell phenotype is to use S2RM technology to reestablish a normal ECM microenvironment for the cancer cell. That is, using one progenitor cell type to release the building blocks of the ECM, such as collagen, and using another stem cell type to release other building blocks and the instruction sets to build the architecture of the ECM, the normal state of the ECM can be rebuilt and lead to the reversion of the cancer cell phenotype to a more normal somatic cell phenotype as depicted in Figure 2.

In summary, the S2RM technology provides a natural means for mimicking and stimulating the healing properties of the human body. Instead of using foreign molecules, natural molecules are used that will induce the initiation of natural processes with little or no side-effects. Further, instead of using a small molecule approach where one molecules interacting at one pathway underlying a multi-pathway disease is used, here the S2RM approach uses multiple molecules to perturb multiple pathways underlying the disease, thus yielding a more efficacious result than the one molecule-one pathway reductionist approach.

The S2RM approach will introduce all of the needed molecules to the tissue to induce a full wound healing cascade of events, unlike an approach using the molecules from one stem cell type that will introduce only a portion of the needed molecules and thus provide a fraction of the efficacy that the S2RM provides. And, S2RM uses the particular molecules from the particular stem cells types relevant to the particular tissue to be healed. This is distinct from the “one size fits all” approach where one stem cell type is used to develop therapeutics for the whole body. Therefore, S2RM provides all of the building blocks, such as the different collagen types, to rebuild the tissue, and also provides the instruction set molecules, such as microRNA, that will deliver the needed architectural commands that will lay the building blocks in their proper places for that particular tissue. During this rebuilding process, the immune response will also be modulated by S2RM, so that inflammation is quelled, allowing the rebuilding to proceed within a normalized framework that is not swollen. The S2RM rebuilding process institutes two fundamental stem cell healing processes: 1. Mimicking the actions of multiple stem cell types and the molecules that they release in the relevant tissue, and 2. Reconditioning the stem cell niche itself and driving the niche to a more primordial, potent state, allowing endogenous stem cell processes to better induce a healing response. Thus, a systems therapeutic approach using multiple molecules from multiple stem cell types called S2RM is used to develop a safer, more natural, and more efficacious therapeutic that both mimics and facilitates the natural adult stem cell healing processes of our body.

FIG. 1. General Model Of Wound Healing

FIG. 1. General Model Of Wound Healing

FIG. 1. The wounded state sends a homing signal to bone marrow stem cells and disrupts the ECM. Disruption of the ECM will shift the dynamic transition of potency towards dedifferentiation and the more pluripotent state. The more pluripotent state will cause the cells to proliferate. After proliferation, the migration of bone marrow stem cells to the wound site will release SRM, including GDF-11, that stops proliferation and induces differentiation allowing newly differentiated somatic cells to repair the tissue. Thus, in our model, GDF11 is released from BMSCs and is a master regulator of stem cell transcription that inhibits cell proliferation and migration by down-regulating the expression of numerous genes involved in both these processes (Williams et al, 2013). ECM-D = Extracellular Matrix Disruption. SC = somatic cell. PPSC = pluripotent stem cell. BMSC = bone marrow stem cell. PSC = potent stem cell.

FIG 2. Regulation Of The Cancer/Pluripotent Phenotype By Stem Cells And Extracellular Matrix

FIG 2. Regulation Of The Cancer/Pluripotent Phenotype By Stem Cells And Extracellular Matrix

FIG. 2. The cancer/pluripotent cell phenotype can be regulated by the ECM and stem cells, where cancer cells can be removed from a dysregulated ECM and placed into a normal ECM and the cancer/pluripotent phenotype will revert to a normal, somatic cell phenotype. Likewise, if a dysregulated ECM is reconstructed into a normal state, the cancer/pluripotent phenotype will revert to the normal somatic cell phenotype. Further regulation of the cancer/pluripotent phenotype can be regulated by a number of factors, including microRNA contained within exosomes that were released from mesenchymal stem cells serving to change the state of the cancer cell into one of quiescence.

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Dr. Greg Maguire, Ph.D.

The SRM Molecular Foundry at UCSD; BioRegenerative Sciences, Inc.

Contact: gmaguire@srmfoundry.org

Maturing From Embryonic To Adult Policy On Stem Cell Therapeutics

Last month, 28 March 2014, the National Institutes of Health (NIH) closed the agency’s Center for Regenerative Medicine (CRM), and the center’s director Dr. Mahendra Rao, a prominent stem cell biologist, left the NIH1. The CRM was established in 2010 to centralize stem cell research activities within the NIH, with the goal to develop therapeutics based on using induced pluripotent stem cells (iPS). The iPS is a mature cell that has been genetically modified, similar to that which is familiar to many people as a genetically modified organism (GMO), to transform the mature cell into a cell with stem cell-like properties. The genetic reprogramming of the mature cell into an iPS means that the newly transformed cell will have properties like an embryonic stem cell whereby the iPS can mature (differentiate) into many types of new cells, whether that new cell type be a nervous system cell or a heart cell or some other cell type, in order to generate that particular tissue in the nervous system or the heart and thus repair the damaged tissue of that particular organ. The importance of the iSC was not only for ethical and religious reasons because an embryo is not destroyed in the making of an iSC, but also because the iSC can be created from somatic cells taken from the same patient that will receive the iSC transplant.

Because the iSC comes from the same donor, the possibility of immune mediated implant rejection is obviated or minimized. The goals of the CRM to focus on the iPS were very ambitious and of great potential importance. But perhaps the goal to focus mainly on iPSs was too narrow. Over the last few year several laboratories have reported reprogramming errors in the iPSs, including genetic errors found by Goldstein’s lab at the University of California, San Diego,2 and epigenetic errors found by Ecker’s lab at the Salk Institute.3 The differences (errors) observed between iPSs and embryonic stem cells fall into the categories of gene copy number variation, chromosome duplication, epigenetic variation, and acquired protein coding point mutations. This means that the fundamental nature of the iPS, and the constituent parts of the cell being formed contain errors and the iPS does