- Email: [email protected]
Stem Cell Released Molecules and Exosomes in Tissue Engineering
Available online at www.sciencedirect.com
Procedia Engineering 59 (2013) 270 – 278
3rd International Conference on Tissue Engineering, ICTE2013
Stem cell released molecules and exosomes in tissue engineering Greg Maguire*, Peter Friedman, Debra McCarthy, Rita Friedman, Andrew Maniotis BioRegnerative Sciences, Inc. 2658 Del Mar Heights Rd #416, San Diego, CA 92014, USA
Abstract Stem cell released molecules (SRM) production employs a proprietary manufacturing process that allows the collection of a wide variety of soluble and properly folded proteins and signaling molecules with complete post-translational modifications and exosome packaging from multiple stem cell types important to immune modulation, and tissue repair and regeneration. The advantages of the process are in the production of biologically active proteins, exosomes, and signaling molecules, developing a “systems therapeutic” yielding a combination of many molecules, that act at multiple targets, resulting in a synergistic therapeutic with emergent therapeutic value. Furthermore, the production of the SRM (stem cell released molecules) does not require downstream solubilization, refolding, or other processes. Additionally, the process offers reduced purification requirements and lower production costs than other pharmacological and biological processes. The production of a “systems therapeutic” with a multitude of molecules represents a multi-targeted, systems biology approach to designing and manufacturing therapeutics, including therapeutics designed to work alone, or augment scaffolding-based and/or cell-based regenerative medicine. © 2013 The Authors. Published by Elsevier Ltd. Selection and peer-review under responsibility of the Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Centro Empresarial da Marinha Grande. Keywords: Stem Cells, SRM, exosomes, systems therapeutic, SRM tissue engineering.
* Corresponding author. Tel.: 001.858.413.7372; fax: 001.877.892.9995. E-mail address: [email protected]
1877-7058 © 2013 The Authors. Published by Elsevier Ltd. Selection and peer-review under responsibility of the Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Centro Empresarial da Marinha Grande doi:10.1016/j.proeng.2013.05.121
Greg Maguire et al. / Procedia Engineering 59 (2013) 270 – 278
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1. Introduction Stem cell released molecules (SRM) represent a “systems therapeutic” where a multitude of molecules released from stem cells, including adult stem cells, act through a variety of mechanisms to induce repair and regeneration of tissue [1]. Indeed, the SRM developed as a “systems therapeutic” is bio-inspired and mimics the endogenous stem cell healing in the human body [2]. Because up to 80% of the therapeutic value of adult stem cells in the human body is through the release of SRM, as opposed to differentiation of the stem cell into mature tissue [3], SRM technology is valuable when used alone as a therapeutic, or when used in conjunction with cell-based and/or scaffolding-based therapeutics. 2. S2RM Demonstrated in recent years has been that two or more stem cell types home into the damaged tissue where each cell type releases a particular pool of SRM into the target site to induce healing. Because each stem cell type releases a unique, but often, overlapping pool of SRM, the two or more pools of SRM acting in a synergistic manner has been described as S2RM [1]. The S2RM technology represents a new direction in the development of therapeutics, distinct from traditional reductionist methods where one small molecule is developed to interact with one primary target pathway [4]. Indeed, reverse engineering led to the S2RM technology where natural stem cell healing processes are mimicked such that a multitude of target pathways underlying the particular indication can be identified and perturbed for amelioration by the molecules.
Figure 1. S2RM is the stem cell released molecules from two or more types of stem cells. SRM from pool 1 combines with SRM from pool 2 to form a synergistic combination pool of SRM termed S2RM. The pool of S2RM contains a fraction of exosomes.
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3. Exosomes Exosomes from stem cells are naturally occurring nanospheres composed of a lipid bilayer, much like a liposome. Important to the exosome is their ability to very efficiently protect, transport, and deliver their contents, which is a wide variety of molecules (SRM), to surrounding cells. The exosome is naturally produced by adult stem cells in culture if the stem cells are properly processed through to final secretion of the SRM that contains the exosomes. That is, collecting the secretome, instead of the lysate, is critical to proper production of SRM and exosomes. Exosomes are especially important for the development of biologics and drug delivery for the aforementioned reasons, and also because the exosomes from stem cells is immunologically inert [5], and can pass through the blood-brain barrier [6]. This will be more thoroughly described in a later section. 4. Stem cell S2RM processing The processing of stem cells for the production of S2RM and exosomes requires a number of important steps. First, the stem cell types relevant to the particular tissue and to the indication are identified. Second, each cell type must be cultured and stimulated in a manner to best elicit the SRM.
Figure 2A (Above). Stem cells derived from ileum. 2B (Below). Exosomes secreted from an adult stem cell are shown in dark-field microscopy as the vast number of white dots in the upper regions of the micrograph...
Third, the stem cells must be allowed to fully process their molecules to the point of secretion into the extracellular space, and fourth, the molecules and exosomes must be collected.
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4.1. S2RM versus other processing technologies The S2RM processing technology for the development of therapeutics is significantly different from other technologies in a number of ways. As we can contrast in Figure 3, S2RM secretome processing has a number of advantages over other technologies. First, S2RM possesses all of the molecules that are relevant and needed for tissue repair and regeneration, whereas other technologies only contain a fraction of the needed molecules. Second, because the secretome is utilized instead of a lysate, the molecules of the SRM have been allowed to complete their post-translational modifications (PTM), whereas other technologies will use molecules that have not completed PTM and are therefore dysfunctional because of mis-folding and improper moiety formation. Third, the secretome processing allows for the complete molecular packaging of the molecules into exosomes for protection, transport, and delivery to neighboring cells. Incomplete molecular packaging can leave the molecules unprotected, poorly transportable, and without efficient delivery to neighboring cells. And, fourth, because native stem cells instead of quasi-stem cells such as iPSCs and parthenogenetic stem cells are used in S2RM, no genetic or epi-genetic programming errors are introduced into the cell’s mechanisms for producing the S2RM. In contradistinction to adult stem cells used in S2RM processing, the quasi-stem cells suffer from many genetic and epi-genetic programming errors rendering a cell type that is severely dysfunctional and producing an incomplete set of molecules [7,8,9,10].
Figure 3. S2RM secretome processing offers a number of advantages for producing therapeutics compared to other techniques.
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4.2. Exosome processing Exosomes from stem cells have been concentrated and separated using a proprietary, patent-pending technology developed at BioRegenerative Sciences, Inc. When fully developed and processed for concentration and collection, the exosomes will be replete with many signaling molecules including growth factors, microRNA, and proteasomes for protein debris removal [11]. 4.2.1. Exosomes from ileum immune system Developing an immune system-derived exosome preparation, our experimental results show that in all analyses of ileum, the production of lineage negative, sca-1-positive, and cKIT positive cells far exceeds the best results obtained from bone marrow. Empirical findings suggest that the natural matrix of the ileum is inhibitory to the augmentation of these putative stem cells in semi-solid media, and that further mincing of the matrix-stripped explants yields unprecedented quantities of lineage-/sca-1+/cKIT+ cells. The patent uses a process whereby buffers dissolve the matrix that encase the exosomes, and the exosomes are then easily collected. Taking care to hydrate the culture chambers can provide months of continuous, sterile, expansion of cells targeted by our strategy-the cells that continue to proliferate the most In Vitro, and by definition, have the most uncommitted lineage-generating potential. Survival of the cultured ileum derived stem cells under defined culture conditions results in a distinct, spatially and temporally patterned release of exosomes from the ileum derived stem cells (IDSCs). When the release of the exosomes is into a specially constructed thickened, semi-solid media that surrounds the stem cells, the physical isolation of the exosomes is achieved by simple extraction methods of that portion of the media containing the exosomes. 5. Developing the artificial stem cell niche Adult stem cells exists throughout most of the adult body, and exists within a very complex, specialized compartment called the stem cell niche [12]. Systemic or bolus injections of S2RM into tissue alone in some ways mimic the niche in providing homing, survival, and proliferation signaling [1,12]. To build or repair missing or highly damaged tissue, scaffolding and/or stem cells may be needed in addition to the S2RM. The scaffolding can provide many signalling substrates not otherwise present. For example, mechanical signal transduction can be so profound that mechanical forces exerted through the cell’s plasma membrane directly to the nucleus can change DNA expression [13,14], and rapidly change stem cell function [15]. As such, the physical and biochemical parameters of the scaffold can be regulated under static and/or dynamic control. For example, a force f will strain any physically linked protein and affect the kinetic rate k of a protein-protein interaction or conformation change as:
k~koexp(f/fo)
(1)
Indeed, stem cells normally depend on an elastic matrix for many functions [16], and may possess more than the typical ensemble of force-coupled signalling pathways as a means to sensitize themselves to microenvironments that range in the physical dimension from flowing fluids and strained tissues to solid tissues of varied elasticity [17], and the numerous physical changes in tissues associated with disease and trauma.
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Figure 4. Scaffolding composed of biopolymer and S2RM produces a matrix with stem cell attractant, and other signaling properties thus producing a quasi-natural tissue construct to build and repair damaged tissues and organs.
Recent advances in dynamic scaffold design include incorporating various polymers into porous foams that shift in stiffness, and controlling the surface topology of the pores and thus the distribution and size of the places where cells and proteins attach in three dimensional space [18]. This technique could allow bioengineers to create selfassembling scaffolds that control, for example, where signaling molecules and stem cells adhere, and under what tensional and/or biochemical conditions the adherence will be self-implemented. Matrix substrates and alignments [19] and biochemical composition [20] will also play key roles for the instruction of stem cell function and tissue formation. For example, electrospun nanofibers of particular physical properties and alignments can preferentially differentiate neural stem cells into Schwann cells [19], and the addition of SRM into the niche or artificial niche will induce the migration of multiple somatic cell types [1,20]. While the importance of understanding the components of the multifactorial stem cell niche is critical to determining stem cell fate [21], equally important is determining the multifactorial nature of controlling the stem cell released molecules [1]. This is important because of the huge therapeutic benefit, up to 80%, of the SRM compared to stem cell fate (differentiation) in adult stem cells responsible for maintenance and repair of our bodies. As shown in Fig. 4, a smart scaffold can be combined with embedded S2RM to attract native stem cells to the scaffold, with the S2RM to help maintain and embed the stem cells in the matrix. The S2RM also provides signalling, including migration and mitotic signalling, to neighbouring cells to enhance the formation of the artificial niche. Of course the scaffold can also be preloaded with stem cells, and the S2RM, to make the artificial tissue or niche. Interestingly, non-scaffolding means of controlling physical parameters of cells, matrix interactions, and juxtaposition signalling can be developed through engineered microsphere technologies [22]. In such a manner, SRM and differentiation parameters can be controlled in circulating or injected stem cells without the need to limit the cells to an interaction with a more physically constrained scaffold. 6. Therapeutic benefits: past, present, and future While the private practice of stem cell-based medicine has sometimes put the “medical cart in front of the scientific horse,” mainstream medical stem cell therapy has been practiced for decades in the form of bone marrow
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tissue transplants. In 1957, stem cell therapeutics made a major stake in medicine with the advent of intravenous infusion of bone marrow in cancer patients [23]. Now using purified stem cells rather than whole bone marrow taken from the patient before chemotherapy, physicians avoid re-injecting patients with their own cancer cells. Today, especially in academic medical centers throughout the world, we witness an array of new therapeutic procedures involving stem cells. For example promising results for Type 1 diabetes has been reported [24]. Stem cells have also been used clinically for bone tissue repair in osteogenesis imperfecta [25], for promotion of tissue regeneration in myocardial infarction, and as immune modulators in the treatment of graft-versus host disease (GvHD) [26,27]. Organ transplant patients are now benefiting from stem cell transplantation as the stem cells reset the immune system and prevent organ rejection by replacing the deadly immunosuppressive drugs normally given to organ transplant patients [28]. SRM technologies as therapeutics have been on the market for years. For example, SRM from a type of progenitor cell has been used in an artificial skin matrix to help close diabetic foot ulcers [29], and S2RM has been used to treat a number of conditions, where the cells necessary for the best therapeutic effect must be carefully considered [1,30]. Like the promise of gene-splicing in the 1970s, which first delivered human insulin as a commercially viable product in 1976 and started the biotechnology industry [31], the power of stem cell technology is so great that an equivalent impact will be felt in society in the next decade. 6.1. Example: Treating immune related conditions Let’s consider an example. Mesenchymal stem cells (MSCs) are immunosuppressive and have been used to treat rejection of organ transplants [28]. Numerous characteristics contribute to the effect. Besides being characterized by low expression of Major Histocompatibility Complex class II (MHCII) and co-stimulatory molecules (B7-1 and B7-2), MSCs interfere with various pathways of the immune response by means of cell-tocell interactions and SRM, including members of the transforming growth factor- family, interleukins 6 and 10, proteasomes, matrix metalloproteinases (MMPs), nitric oxide and indoleamine 2,3 deoxygenase (IDO). Different studies have reported the ability of MSCs to suppress T-cell proliferation, most likely via Prostaglandin E2 (PGE2) production [32], to induce T regulatory cells [33], and to express co-inhibitory molecules as B7-H1 on their surface upon IFN- treatment [34]. Further, MSCs can impair maturation and function of dendritic cells and inhibit the proliferation, differentiation, and chemotaxis of B-cells in vitro [35]. The immune-stimulating properties of these adult stem cells have been reported in some studies and may depend on the production of pro-inflammatory cytokines [36]. Evidence suggests that dual immunoregulatory function of MSCs is dose-dependent, because high numbers of MSCs suppress whereas very low numbers seem to stimulate lymphocyte proliferation [37]. Dose dependency has important implications in the use of MSCs as celltherapeutics, as the dosing schedule is likely critical for the in vivo function and may rely on factors that are not well-understood, thereby limiting widespread use in the clinic. Such problems for stem cells may be mitigated by using the S2RM instead of the cells where possible immune stimulation is eliminated or mitigated, and where dosing of the molecules can be under much better control in space and time. Here too, the smart scaffolding may play a role in releasing the S2RM/exosomes from the scaffolds defined physical dimension, while also potentially releasing the molecules on a demand basis, for example, when the scaffold senses hypoxic conditions or a physical change such as swelling. 6.2. Stem cell therapeutics without cells Of great importance to the advancement of stem cell-based therapeutics are the observations that exosomes are immunoprivileged [5] and that ongoing MSC-based trials for treatment of disease, including cardiovascular diseases as an example, reveal an interesting trend in clinical trial designs, in that SRM mechanisms for improving angiogenesis, cardio-myogenesis, stimulating endogenous cardiac progenitors and inhibiting remodelling have been highlighted as the primary mode of action [38]. Inflammation underlies a number of diseases and other indications, and recent studies show that many conditions involving inflammation can be successfully treated with stem cells, including asthma [39], where S2RM has been shown to be helpful (author’s observation). More and
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more studies report that the therapeutic effect of stem cells can be largely attributed to their SRM [3,40]. Thus, the current research portends an advantage to systems biology-based S2RM and exosome methodologies when used alone, or in conjunction with cell technologies and/or scaffolding technologies for the development of therapeutics to address many indications. Considering the blind men and the elephant parable from ancient India, if we choose to consider disease and other indications in a reductionist manner as involving one leg, or one tail, and then developing a therapeutic to treat that one pathway, the leg, or the tail, we will fail to recognize the elephant and therefore neither recognize the condition nor provide the best treatment. Reverse engineering natural stem cell repair and regeneration mechanisms, where the stem cells have evolved to recognize the many pathways of the elephant, and thus the elephant becomes an emergent property of the system, leads to better therapeutics through the development of stem cell-based bioinspired “systems therapeutics.” Acknowledgements We thank Mr. Stewie and Mr. Mambo for their early contributions to prototyping our technology. References [1] Maguire, G., Friedman, P., 2013. The systems biology of stem cell released molecules-based therapeutics. .ISRN Stem Cells. V 2013 12 pages. [2] Maguire, G., Friedman, P., 2013. Enhancing spontaneous stem cell healing. Journal of Complementary and Integrative Medicine, In Press. [3] Chimenti, I., et al. 2010. Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice. Circulation Research 106: 971-980. [4] Maguire, G. 2013. Using a systems-based approach for the development of diagnostics. Expert Review of Molecular Diagnostics, In Press. [5] Lai, RC, Yeo, RW, Tan, KH, Lim, SK. 2012. Exosomes for drug delivery – a novel application for the mesenchymal stem cell. Biotecnol. Adv. [6] Alvarez-Ervit, L. et al. 2011. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nature Biotech. 29: 341345. [7] Mayshar, Y. et al. 2010 Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell 7: 521-531. [8] Lister, R. 2011. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature. 3; 471(7336): 68–73. [9] Gore, a. et al. 2011 Somatic coding mutations in human induced pluripotent stem cells. Nature. Nature. 2011 March 3; 471(7336): 63–67. [10] Naturil-Alfonso, C. et al. 2012. Transcriptome Profiling of Rabbit Parthenogenetic Blastocysts Developed under In Vivo Conditions. PLoS One. 2012; 7(12): e51271. [11] Lai, R.c. et al 2012. Proteolytic potential of MSC exosome proteome: implications for exosome-mediated delivery of therapeutic proteasome. [12] Scadden, D.T. 2006. The stem cell niche as an entity of action. Nature 441: 1075-1079. [13] Maniotis, A., Chen,C.S., Ingber, D.E. 1997 Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):849-54. [14] Milstein, J.N., Meiners, J.C. 2011. On the role of DNA biomechanics in the regulation of gene expression. J. Roy. Soc. Interface. doi: 10.1098/rsif.2011.0371 [15] Sen, B., et al 2010. Mechanical signal inÀuence on mesenchymal stem cell fate is enhanced by incorporation of refractory periods into the loading regimen. doi:10.1016/j.jbiomech.2010.11.022 [16] Engler, A.J. et al. 2006. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell. 126: 677 689. [17] Discher, D.E., Mooney, D.J., Zandstra, P. 2009. Growth factors, matrices, and forces combine and control stem cells. Science. 2009 June 26; 324(5935): 1673–1677. [18] Viswanathan, P. et al. 2012. Cell Instructive Microporous Sca olds through Interface Engineering. J Am Chem Soc 134(49): 20103– 20109. [19] Ren YJ. et al. 2013. Enhanced Differentiation of Human Neural Crest Stem Cells Towards Schwann Cell Lineage by Aligned Electrospun Fiber Matrix. Acta Biomater. 2013 Apr 26. pii: S1742-7061(13)00215-8. doi: 10.1016/j.actbio.2013.04.034. [20] Hu, L. et al. 2013. Effects of adipose stem cell-conditioned medium on the migration of vascular endothelial cells, fibroblasts and keratinocyte. Exp Ther Med. 2013 March; 5(3): 701–706. [21] Gobba, S. et al. 2011. Artificial niche microarrays for probing single stem cell fate in high throughput. Nature Methods 8, 949–955 [22] Sarkar, D., et al. 2012. Cellular and Extracellular Programming of Cell Fate through Engineered Intracrine-, Paracrine-, and Endocrine-like Mechanisms. Biomaterials. 11: 3053–3061.
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[23] Thomas ED, Lochte HL, Jr., Lu WC, Ferrebee JW. 1957. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N Engl J Med.;257:491–496 [24] Zhao, Y. et al, 2012. Reversal of type 1 diabetes via islet cell regeneration following immune modulation by cord blood-derived multipotent stem cells. BMC Med. 2012; 10: 3. [25] Mauney JR, Volloch V, Kaplan DL. 2005. Role of adult mesenchymal stem cells in bone tissue engineering applications: current status and future prospects. Tissue Eng 11: 787–802. [26] Le Blanc K, et al.2004. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 363: 1439–1441. [27] Paul D, Samuel SM, Maulik N. 2009. Mesenchymal stem cell: present challenges and prospective cellular cardiomyoplasty approaches for myocardial regeneration. Antioxid Redox Signal 11: 1841–1855. [28] Scandling, J.D. et al. 2012. Tolerance and Withdrawal of Immunosuppressive Drugs in Patients Given Kidney and Hematopoietic Cell Transplants. Am J Transplant. 12(5): 1133–1145. [29] Warriner, R.A. et al. 2011. Human fibroblast-derived dermal substitute: results from a treatment investigational device exemption (TIDE) study in diabetic foot ulcers. Adv Skin Wound Care. 24(7):306-11. [30] Tao-Sheng, L. et al. 2012. Direct comparison of different stem cell types and subpopulations reveals superior paracrine potency and myocardial repair efficacy with cardiosphere-derived cells. J Am Coll Cardiol. 59(10): 942–953. [31] Russo, E. 2003. Special Report: The Birth of Biotechnology.. Nature. 421, 456-457 [32] Jarvinen et al., 2008. Lung resident mesenchymal stem cells isolated from human lung allografts inhibit T cell proliferation via a soluble mediator. J. Immunol. 181, 4389–4396. [33] Casiraghi F. et al. 2008. Pretransplant infusion of mesenchymal stem cells prolongs the survival of a semiallogeneic heart transplant through the generation of regulatory T cells. J. Immunol. 181: 3933–3946. [34] Sheng H. et al. 2008. A critical role of IFNgamma in priming MSC-mediated suppression of T cell proliferation through up-regulation of B7-H1. Cell Res. 18: 846–857. [35] Corcione, A. et al. 2006. Human mesenchymal stem cells modulate B-cell functions. Blood 107: 367–372. [36] Rasmusson I. 2007. Mesenchymal stem cells stimulate antibody secretion in human B cells. Scand. J. Immunol. 65: 336–343. [37] Le Blanc K. et al. 2003. Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand. J. Immunol. 57, 11–20. [38] Raganath, S.H., et al. 2012. Harnessing the Mesenchymal Stem Cell Secretome for the Treatment of Cardiovascular Disease. Cell Stem Cell. 10: 244–258. [39] Nemeth, K. et al. 2010. Bone marrow stromal cells use TGF- to suppress allergic responses in a mouse model of ragweed-induced asthma. Proc Natl Acad Sci U S A. 107: 5652–5657. [40] Herrera, M.B. et al. 2010. Human liver stem cell-derived microvesicles accelerate hepatic regeneration in hepatectomized rats. J Cell Mol Med. 14: 1605–1618.
Procedia Engineering 59 (2013) 270 – 278
3rd International Conference on Tissue Engineering, ICTE2013
Stem cell released molecules and exosomes in tissue engineering Greg Maguire*, Peter Friedman, Debra McCarthy, Rita Friedman, Andrew Maniotis BioRegnerative Sciences, Inc. 2658 Del Mar Heights Rd #416, San Diego, CA 92014, USA
Abstract Stem cell released molecules (SRM) production employs a proprietary manufacturing process that allows the collection of a wide variety of soluble and properly folded proteins and signaling molecules with complete post-translational modifications and exosome packaging from multiple stem cell types important to immune modulation, and tissue repair and regeneration. The advantages of the process are in the production of biologically active proteins, exosomes, and signaling molecules, developing a “systems therapeutic” yielding a combination of many molecules, that act at multiple targets, resulting in a synergistic therapeutic with emergent therapeutic value. Furthermore, the production of the SRM (stem cell released molecules) does not require downstream solubilization, refolding, or other processes. Additionally, the process offers reduced purification requirements and lower production costs than other pharmacological and biological processes. The production of a “systems therapeutic” with a multitude of molecules represents a multi-targeted, systems biology approach to designing and manufacturing therapeutics, including therapeutics designed to work alone, or augment scaffolding-based and/or cell-based regenerative medicine. © 2013 The Authors. Published by Elsevier Ltd. Selection and peer-review under responsibility of the Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Centro Empresarial da Marinha Grande. Keywords: Stem Cells, SRM, exosomes, systems therapeutic, SRM tissue engineering.
* Corresponding author. Tel.: 001.858.413.7372; fax: 001.877.892.9995. E-mail address: [email protected]
1877-7058 © 2013 The Authors. Published by Elsevier Ltd. Selection and peer-review under responsibility of the Centre for Rapid and Sustainable Product Development, Polytechnic Institute of Leiria, Centro Empresarial da Marinha Grande doi:10.1016/j.proeng.2013.05.121
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1. Introduction Stem cell released molecules (SRM) represent a “systems therapeutic” where a multitude of molecules released from stem cells, including adult stem cells, act through a variety of mechanisms to induce repair and regeneration of tissue [1]. Indeed, the SRM developed as a “systems therapeutic” is bio-inspired and mimics the endogenous stem cell healing in the human body [2]. Because up to 80% of the therapeutic value of adult stem cells in the human body is through the release of SRM, as opposed to differentiation of the stem cell into mature tissue [3], SRM technology is valuable when used alone as a therapeutic, or when used in conjunction with cell-based and/or scaffolding-based therapeutics. 2. S2RM Demonstrated in recent years has been that two or more stem cell types home into the damaged tissue where each cell type releases a particular pool of SRM into the target site to induce healing. Because each stem cell type releases a unique, but often, overlapping pool of SRM, the two or more pools of SRM acting in a synergistic manner has been described as S2RM [1]. The S2RM technology represents a new direction in the development of therapeutics, distinct from traditional reductionist methods where one small molecule is developed to interact with one primary target pathway [4]. Indeed, reverse engineering led to the S2RM technology where natural stem cell healing processes are mimicked such that a multitude of target pathways underlying the particular indication can be identified and perturbed for amelioration by the molecules.
Figure 1. S2RM is the stem cell released molecules from two or more types of stem cells. SRM from pool 1 combines with SRM from pool 2 to form a synergistic combination pool of SRM termed S2RM. The pool of S2RM contains a fraction of exosomes.
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3. Exosomes Exosomes from stem cells are naturally occurring nanospheres composed of a lipid bilayer, much like a liposome. Important to the exosome is their ability to very efficiently protect, transport, and deliver their contents, which is a wide variety of molecules (SRM), to surrounding cells. The exosome is naturally produced by adult stem cells in culture if the stem cells are properly processed through to final secretion of the SRM that contains the exosomes. That is, collecting the secretome, instead of the lysate, is critical to proper production of SRM and exosomes. Exosomes are especially important for the development of biologics and drug delivery for the aforementioned reasons, and also because the exosomes from stem cells is immunologically inert [5], and can pass through the blood-brain barrier [6]. This will be more thoroughly described in a later section. 4. Stem cell S2RM processing The processing of stem cells for the production of S2RM and exosomes requires a number of important steps. First, the stem cell types relevant to the particular tissue and to the indication are identified. Second, each cell type must be cultured and stimulated in a manner to best elicit the SRM.
Figure 2A (Above). Stem cells derived from ileum. 2B (Below). Exosomes secreted from an adult stem cell are shown in dark-field microscopy as the vast number of white dots in the upper regions of the micrograph...
Third, the stem cells must be allowed to fully process their molecules to the point of secretion into the extracellular space, and fourth, the molecules and exosomes must be collected.
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4.1. S2RM versus other processing technologies The S2RM processing technology for the development of therapeutics is significantly different from other technologies in a number of ways. As we can contrast in Figure 3, S2RM secretome processing has a number of advantages over other technologies. First, S2RM possesses all of the molecules that are relevant and needed for tissue repair and regeneration, whereas other technologies only contain a fraction of the needed molecules. Second, because the secretome is utilized instead of a lysate, the molecules of the SRM have been allowed to complete their post-translational modifications (PTM), whereas other technologies will use molecules that have not completed PTM and are therefore dysfunctional because of mis-folding and improper moiety formation. Third, the secretome processing allows for the complete molecular packaging of the molecules into exosomes for protection, transport, and delivery to neighboring cells. Incomplete molecular packaging can leave the molecules unprotected, poorly transportable, and without efficient delivery to neighboring cells. And, fourth, because native stem cells instead of quasi-stem cells such as iPSCs and parthenogenetic stem cells are used in S2RM, no genetic or epi-genetic programming errors are introduced into the cell’s mechanisms for producing the S2RM. In contradistinction to adult stem cells used in S2RM processing, the quasi-stem cells suffer from many genetic and epi-genetic programming errors rendering a cell type that is severely dysfunctional and producing an incomplete set of molecules [7,8,9,10].
Figure 3. S2RM secretome processing offers a number of advantages for producing therapeutics compared to other techniques.
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4.2. Exosome processing Exosomes from stem cells have been concentrated and separated using a proprietary, patent-pending technology developed at BioRegenerative Sciences, Inc. When fully developed and processed for concentration and collection, the exosomes will be replete with many signaling molecules including growth factors, microRNA, and proteasomes for protein debris removal [11]. 4.2.1. Exosomes from ileum immune system Developing an immune system-derived exosome preparation, our experimental results show that in all analyses of ileum, the production of lineage negative, sca-1-positive, and cKIT positive cells far exceeds the best results obtained from bone marrow. Empirical findings suggest that the natural matrix of the ileum is inhibitory to the augmentation of these putative stem cells in semi-solid media, and that further mincing of the matrix-stripped explants yields unprecedented quantities of lineage-/sca-1+/cKIT+ cells. The patent uses a process whereby buffers dissolve the matrix that encase the exosomes, and the exosomes are then easily collected. Taking care to hydrate the culture chambers can provide months of continuous, sterile, expansion of cells targeted by our strategy-the cells that continue to proliferate the most In Vitro, and by definition, have the most uncommitted lineage-generating potential. Survival of the cultured ileum derived stem cells under defined culture conditions results in a distinct, spatially and temporally patterned release of exosomes from the ileum derived stem cells (IDSCs). When the release of the exosomes is into a specially constructed thickened, semi-solid media that surrounds the stem cells, the physical isolation of the exosomes is achieved by simple extraction methods of that portion of the media containing the exosomes. 5. Developing the artificial stem cell niche Adult stem cells exists throughout most of the adult body, and exists within a very complex, specialized compartment called the stem cell niche [12]. Systemic or bolus injections of S2RM into tissue alone in some ways mimic the niche in providing homing, survival, and proliferation signaling [1,12]. To build or repair missing or highly damaged tissue, scaffolding and/or stem cells may be needed in addition to the S2RM. The scaffolding can provide many signalling substrates not otherwise present. For example, mechanical signal transduction can be so profound that mechanical forces exerted through the cell’s plasma membrane directly to the nucleus can change DNA expression [13,14], and rapidly change stem cell function [15]. As such, the physical and biochemical parameters of the scaffold can be regulated under static and/or dynamic control. For example, a force f will strain any physically linked protein and affect the kinetic rate k of a protein-protein interaction or conformation change as:
k~koexp(f/fo)
(1)
Indeed, stem cells normally depend on an elastic matrix for many functions [16], and may possess more than the typical ensemble of force-coupled signalling pathways as a means to sensitize themselves to microenvironments that range in the physical dimension from flowing fluids and strained tissues to solid tissues of varied elasticity [17], and the numerous physical changes in tissues associated with disease and trauma.
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Figure 4. Scaffolding composed of biopolymer and S2RM produces a matrix with stem cell attractant, and other signaling properties thus producing a quasi-natural tissue construct to build and repair damaged tissues and organs.
Recent advances in dynamic scaffold design include incorporating various polymers into porous foams that shift in stiffness, and controlling the surface topology of the pores and thus the distribution and size of the places where cells and proteins attach in three dimensional space [18]. This technique could allow bioengineers to create selfassembling scaffolds that control, for example, where signaling molecules and stem cells adhere, and under what tensional and/or biochemical conditions the adherence will be self-implemented. Matrix substrates and alignments [19] and biochemical composition [20] will also play key roles for the instruction of stem cell function and tissue formation. For example, electrospun nanofibers of particular physical properties and alignments can preferentially differentiate neural stem cells into Schwann cells [19], and the addition of SRM into the niche or artificial niche will induce the migration of multiple somatic cell types [1,20]. While the importance of understanding the components of the multifactorial stem cell niche is critical to determining stem cell fate [21], equally important is determining the multifactorial nature of controlling the stem cell released molecules [1]. This is important because of the huge therapeutic benefit, up to 80%, of the SRM compared to stem cell fate (differentiation) in adult stem cells responsible for maintenance and repair of our bodies. As shown in Fig. 4, a smart scaffold can be combined with embedded S2RM to attract native stem cells to the scaffold, with the S2RM to help maintain and embed the stem cells in the matrix. The S2RM also provides signalling, including migration and mitotic signalling, to neighbouring cells to enhance the formation of the artificial niche. Of course the scaffold can also be preloaded with stem cells, and the S2RM, to make the artificial tissue or niche. Interestingly, non-scaffolding means of controlling physical parameters of cells, matrix interactions, and juxtaposition signalling can be developed through engineered microsphere technologies [22]. In such a manner, SRM and differentiation parameters can be controlled in circulating or injected stem cells without the need to limit the cells to an interaction with a more physically constrained scaffold. 6. Therapeutic benefits: past, present, and future While the private practice of stem cell-based medicine has sometimes put the “medical cart in front of the scientific horse,” mainstream medical stem cell therapy has been practiced for decades in the form of bone marrow
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tissue transplants. In 1957, stem cell therapeutics made a major stake in medicine with the advent of intravenous infusion of bone marrow in cancer patients [23]. Now using purified stem cells rather than whole bone marrow taken from the patient before chemotherapy, physicians avoid re-injecting patients with their own cancer cells. Today, especially in academic medical centers throughout the world, we witness an array of new therapeutic procedures involving stem cells. For example promising results for Type 1 diabetes has been reported [24]. Stem cells have also been used clinically for bone tissue repair in osteogenesis imperfecta [25], for promotion of tissue regeneration in myocardial infarction, and as immune modulators in the treatment of graft-versus host disease (GvHD) [26,27]. Organ transplant patients are now benefiting from stem cell transplantation as the stem cells reset the immune system and prevent organ rejection by replacing the deadly immunosuppressive drugs normally given to organ transplant patients [28]. SRM technologies as therapeutics have been on the market for years. For example, SRM from a type of progenitor cell has been used in an artificial skin matrix to help close diabetic foot ulcers [29], and S2RM has been used to treat a number of conditions, where the cells necessary for the best therapeutic effect must be carefully considered [1,30]. Like the promise of gene-splicing in the 1970s, which first delivered human insulin as a commercially viable product in 1976 and started the biotechnology industry [31], the power of stem cell technology is so great that an equivalent impact will be felt in society in the next decade. 6.1. Example: Treating immune related conditions Let’s consider an example. Mesenchymal stem cells (MSCs) are immunosuppressive and have been used to treat rejection of organ transplants [28]. Numerous characteristics contribute to the effect. Besides being characterized by low expression of Major Histocompatibility Complex class II (MHCII) and co-stimulatory molecules (B7-1 and B7-2), MSCs interfere with various pathways of the immune response by means of cell-tocell interactions and SRM, including members of the transforming growth factor- family, interleukins 6 and 10, proteasomes, matrix metalloproteinases (MMPs), nitric oxide and indoleamine 2,3 deoxygenase (IDO). Different studies have reported the ability of MSCs to suppress T-cell proliferation, most likely via Prostaglandin E2 (PGE2) production [32], to induce T regulatory cells [33], and to express co-inhibitory molecules as B7-H1 on their surface upon IFN- treatment [34]. Further, MSCs can impair maturation and function of dendritic cells and inhibit the proliferation, differentiation, and chemotaxis of B-cells in vitro [35]. The immune-stimulating properties of these adult stem cells have been reported in some studies and may depend on the production of pro-inflammatory cytokines [36]. Evidence suggests that dual immunoregulatory function of MSCs is dose-dependent, because high numbers of MSCs suppress whereas very low numbers seem to stimulate lymphocyte proliferation [37]. Dose dependency has important implications in the use of MSCs as celltherapeutics, as the dosing schedule is likely critical for the in vivo function and may rely on factors that are not well-understood, thereby limiting widespread use in the clinic. Such problems for stem cells may be mitigated by using the S2RM instead of the cells where possible immune stimulation is eliminated or mitigated, and where dosing of the molecules can be under much better control in space and time. Here too, the smart scaffolding may play a role in releasing the S2RM/exosomes from the scaffolds defined physical dimension, while also potentially releasing the molecules on a demand basis, for example, when the scaffold senses hypoxic conditions or a physical change such as swelling. 6.2. Stem cell therapeutics without cells Of great importance to the advancement of stem cell-based therapeutics are the observations that exosomes are immunoprivileged [5] and that ongoing MSC-based trials for treatment of disease, including cardiovascular diseases as an example, reveal an interesting trend in clinical trial designs, in that SRM mechanisms for improving angiogenesis, cardio-myogenesis, stimulating endogenous cardiac progenitors and inhibiting remodelling have been highlighted as the primary mode of action [38]. Inflammation underlies a number of diseases and other indications, and recent studies show that many conditions involving inflammation can be successfully treated with stem cells, including asthma [39], where S2RM has been shown to be helpful (author’s observation). More and
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more studies report that the therapeutic effect of stem cells can be largely attributed to their SRM [3,40]. Thus, the current research portends an advantage to systems biology-based S2RM and exosome methodologies when used alone, or in conjunction with cell technologies and/or scaffolding technologies for the development of therapeutics to address many indications. Considering the blind men and the elephant parable from ancient India, if we choose to consider disease and other indications in a reductionist manner as involving one leg, or one tail, and then developing a therapeutic to treat that one pathway, the leg, or the tail, we will fail to recognize the elephant and therefore neither recognize the condition nor provide the best treatment. Reverse engineering natural stem cell repair and regeneration mechanisms, where the stem cells have evolved to recognize the many pathways of the elephant, and thus the elephant becomes an emergent property of the system, leads to better therapeutics through the development of stem cell-based bioinspired “systems therapeutics.” Acknowledgements We thank Mr. Stewie and Mr. Mambo for their early contributions to prototyping our technology. References [1] Maguire, G., Friedman, P., 2013. The systems biology of stem cell released molecules-based therapeutics. .ISRN Stem Cells. V 2013 12 pages. [2] Maguire, G., Friedman, P., 2013. Enhancing spontaneous stem cell healing. Journal of Complementary and Integrative Medicine, In Press. [3] Chimenti, I., et al. 2010. Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice. Circulation Research 106: 971-980. [4] Maguire, G. 2013. Using a systems-based approach for the development of diagnostics. Expert Review of Molecular Diagnostics, In Press. [5] Lai, RC, Yeo, RW, Tan, KH, Lim, SK. 2012. Exosomes for drug delivery – a novel application for the mesenchymal stem cell. Biotecnol. Adv. [6] Alvarez-Ervit, L. et al. 2011. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nature Biotech. 29: 341345. [7] Mayshar, Y. et al. 2010 Identification and classification of chromosomal aberrations in human induced pluripotent stem cells. Cell Stem Cell 7: 521-531. [8] Lister, R. 2011. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature. 3; 471(7336): 68–73. [9] Gore, a. et al. 2011 Somatic coding mutations in human induced pluripotent stem cells. Nature. Nature. 2011 March 3; 471(7336): 63–67. [10] Naturil-Alfonso, C. et al. 2012. Transcriptome Profiling of Rabbit Parthenogenetic Blastocysts Developed under In Vivo Conditions. PLoS One. 2012; 7(12): e51271. [11] Lai, R.c. et al 2012. Proteolytic potential of MSC exosome proteome: implications for exosome-mediated delivery of therapeutic proteasome. [12] Scadden, D.T. 2006. The stem cell niche as an entity of action. Nature 441: 1075-1079. [13] Maniotis, A., Chen,C.S., Ingber, D.E. 1997 Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):849-54. [14] Milstein, J.N., Meiners, J.C. 2011. On the role of DNA biomechanics in the regulation of gene expression. J. Roy. Soc. Interface. doi: 10.1098/rsif.2011.0371 [15] Sen, B., et al 2010. Mechanical signal inÀuence on mesenchymal stem cell fate is enhanced by incorporation of refractory periods into the loading regimen. doi:10.1016/j.jbiomech.2010.11.022 [16] Engler, A.J. et al. 2006. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell. 126: 677 689. [17] Discher, D.E., Mooney, D.J., Zandstra, P. 2009. Growth factors, matrices, and forces combine and control stem cells. Science. 2009 June 26; 324(5935): 1673–1677. [18] Viswanathan, P. et al. 2012. Cell Instructive Microporous Sca olds through Interface Engineering. J Am Chem Soc 134(49): 20103– 20109. [19] Ren YJ. et al. 2013. Enhanced Differentiation of Human Neural Crest Stem Cells Towards Schwann Cell Lineage by Aligned Electrospun Fiber Matrix. Acta Biomater. 2013 Apr 26. pii: S1742-7061(13)00215-8. doi: 10.1016/j.actbio.2013.04.034. [20] Hu, L. et al. 2013. Effects of adipose stem cell-conditioned medium on the migration of vascular endothelial cells, fibroblasts and keratinocyte. Exp Ther Med. 2013 March; 5(3): 701–706. [21] Gobba, S. et al. 2011. Artificial niche microarrays for probing single stem cell fate in high throughput. Nature Methods 8, 949–955 [22] Sarkar, D., et al. 2012. Cellular and Extracellular Programming of Cell Fate through Engineered Intracrine-, Paracrine-, and Endocrine-like Mechanisms. Biomaterials. 11: 3053–3061.
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