“The reports of my death are greatly exaggerated”
The text of a cable sent by Mark Twain in response to the mistakenly published press release with his obituary.
Failed organs, according to the well established doctrine, can be substituted by transplanting their analogs obtained from a cadaveric or a live donor. Success of this approach, as remarkable as it is, however, is restricted by the limited volume of available donors of either type. A more recent alternative way of thinking focuses on therapeutic regeneration, which is commonly achieved by either genome editing and gene therapy, stem/progenitor cell therapy and transplantation, or organ bioengineering utilizing stem or induced pluripotent stem cells. These technically complex and, at times, ethically ambiguous therapeutic modalities are directed to repair or substitute a damaged organ or a cell type. Gains and promises along these lines of investigation have been exhaustively reviewed. The above approaches are on the basis of substitutive therapies of failed cells or organs, with the conceptual underpinning of finality in failed differentiated cells. These strategies in regenerative medicine have been thoroughly and critically reviewed. The number of registered clinical trials (http://clinicaltrials.gov) utilizing stem cells, mesenchymal stem or endothelial progenitor cells of diverse origins, far exceeds 200, with no dramatic successes reported so far.1 Certainly, the marginal progress at this early stage does not disqualify the field of stem cell transplantation for the purposes of regeneration, but it undoubtedly points to the complexity of this strategy.
Lately, the premise of doomsday finality of failed cell fate, just as was the case with Mark Twain’s obituary, has been challenged. Initially, emerging senolytic and rejuvenation therapies have been shown to reverse some failing cell and organ functions (see below). In parallel, the old observations by Gudron’s group2 on the ability of a cell extract to direct differentiation of distinct cells have been put on a broader platform of cell reprogramming. This trend has been followed by most recent developments in the field of cellular reconditioning as the means to restore lost or failed organelles and functions. In the following pages, I will succinctly review these emerging trends in regenerative medicine to highlight their potential and drawbacks.
Ever since the discovery of transcriptional regulators of pluripotency, these factors have been used for generating embryonic-like cells with unlimited differentiation capacity, described by Waddington as reprogramming to pluripotency (reviewed by Srivastava and DeWitt3). Concerns related to tumorigenicity and teratoma formation by these cells hamper their direct applicability. On the other hand, ex vivo forced differentiation of induced pluripotent stem cells to distinct lineages have encountered another problem, namely, cell cycle arrest after differentiation3 and inability to expand such cells for therapeutic transplantation. This gave rise to the search for a direct reprogramming bypassing pluripotent state.
Attempts to change the trajectory of cell differentiation are accumulating (Figure 1). Successful combinatorial approaches for direct conversion of fibroblasts to neuronal cells, cardiomyocytes, and other differentiated cells have been recently summarized in an excellent review by Srivastava and DeWitt.3 Authors emphasize the existing challenges in translating cardiac reprogramming to humans, considering it a work in progress. In the field of fibrotic kidney disease, developmental studies by McMahon and Kobayashi’s groups4 disclosed Six2 and Pax2 as transcription factors for nephron progenitor state and delineation of nephron-interstitium boundaries, thus suggesting a possible blueprint for crossing this boundary in converting myofibroblasts to epithelial cells. Direct in vitro reprogramming of mouse and human fibroblasts into renal tubular epithelia using combined expression of cell fate–specific transcription factors resulted in a successful integration in decellularized kidney scaffolds.5 More recently, an ovo-like Zinc-finger 2 transcription repressor factor has been shown enhance mesenchymal-to-epithelial (keratinocyte) transition.6 Those approaches for direct cell reprogramming should not conflict with the results of in vivo clonal analysis of mammalian kidney, which revealed fate-restricted unipotent segment-specific progenitors involved in tubulogenesis, maintenance, and regeneration.7In vivo administration of mesenchymal stem cells or their conditioned culture medium salvaged kidney function after AKI via a paracrine effect.8 Papadimou et al.9 used a different approach: exposure of transiently permeabilized mesenchymal stem cells to the cellfree extract of renal epithelial cells that resulted in the acquisition of transmonolayer electrical resistance, E-cadherin and aquaporin-1 expression, and brush border microvilli and tight junctions, all convincingly demonstrating that original mesenchymal stem cells were successfully reprogrammed to epithelial cells with properties of the proximal tubular epithelium. In a cisplatin model of AKI, reprogrammed cells integrated into proximal tubule lining and improved renal function. Furthermore, Matsumoto et al.10 attempted to reverse endothelial-to-mesenchymal transition in the course of fibrotic kidney diseases, such as unilateral ureteral obstruction or chronic phase of folic acid–induced nephropathy, using in vitro and in vivo treatment of (myo)fibroblasts with the cellfree extract of endothelial progenitor cells. Exposure of TGF-β–stimulated fibroblasts to the extract halted their conversion to myofibroblasts; similarly, fibrogenesis was attenuated in the above fibrotic models by renal subcapsular injection of the cellfree extract. One of the components of the extract was identified as leukemia inhibitory factor and we found that it is capable of preventing the development of contractile phenotype of activated fibroblasts. It has also induced some pluripotency factors, Nanog and c-Myc, indicative of a partial pluripotency intermediate state. Whether such cells could be transient amplifying progenitors remains to be established. In vivo, a powerful, leukemia inhibitory factor receptor–independent agonist of gp130/STAT3 pathway, hyper–IL-6, prevented renal fibrosis. A similar attempt to reverse endothelial-mesenchymal transition was reported in a model of cardiac fibrosis by Ubil et al.11 These investigators used genetic fate map strategy to show that about one third of fibroblasts in the postischemic heart gained expression of endothelial-specific markers. This process of mesenchymal-to-endothelial transition in the ischemic heart is governed by p53: its loss decreases, whereas its stimulation increases formation of fibroblast-derived endothelial cells, enhances vascular density in the postinfarcted myocardium, and improves cardiac function. Data obtained by Yang et al.12 in mice with unilateral ureteral obstruction showed attenuation of renal fibrosis after blockade of p53.
Studies from Halloran’s laboratory have pioneered the field of premature cell senescence in kidney disease.13 Fogo’s group14 explored effects of transplanted bone marrow–derived cells obtained from young or old mice (8 weeks and 12 months, respectively) on the kidneys of 12-month-old mice. These investigators demonstrated that bone marrow cells obtained from young animals reduced the expression of markers of senescence, reduced collagen deposition, and increased expression of the antiaging gene Klotho. Prospective renal transplant biopsy specimens revealed induction of p21Cip1 in kidneys undergoing AKI-to-CKD continuum.15
In a study of obesity-induced diabetes in mice, Chen et al.16 performed adoptive transfer of syngeneic bone marrow-derived cells harvested from young and old diabetic db/db mice or nondiabetic dbm controls. Diabetic recipients of bone marrow derived from dbm, but not db/db donor mice experienced better glucose control, enhanced insulin sensitivity, striking improvement of endothelium-dependent vasorelaxation in response to acetylcholine, and partially restored renal function. Ex vivo overnight treatment of bone marrow-derived cells from diabetic donors with a selenoorganic antioxidant and peroxynitrite scavenger ebselen, before their adaptive transfer, enabled them to improve vascular and renal functions, similar to the effect of cells from dbm donors. Administration of ebselen in vivo elicited the same effect. These studies represent earlier examples of senescence and premature senescence affecting renal functions and their reversal by instituting rejuvenating therapies. More recently, this field has been dramatically expanded with the direct demonstration that elimination of p16Ink4a-positive senescent cells delays aging-associated disorders.17 Production of proinflammatory and profibrogenic senescence-associated secretory products appears to be primarily responsible for the propagation of the initial lesions, including kidney pathology, failed intrinsic regenerative mechanisms, and progression of chronic disease. Several excellent recent reviews detail these processes occurring in renal disease18,19 and examples of usage of senolytics, like quercetin, in reversing premature kidney senescence and dysfunction.
The idea that normal vasculature maintains structural and functional integrity of surrounding tissues and accelerates their regeneration—angiocrine function of the endothelium—has percolated. Along came the realization that dysfunctional cells are the source of secretory signals, hampering regeneration and instead promoting fibrosis,20 somewhat similar to senescence-associated secretory products; indeed, such cells show increased frequency of premature stress-induced senescence. The corollary of this line of investigations is represented by attempts to manipulate the secretomes of dysfunctional endothelial or epithelial cells to mitigate fibrogenic programs in adjacent fibroblasts (reviewed by Lipphardt et al.20). Such a myofibroblast reprogramming through “reconditioning” of microenvironment represents another emerging trend, as discussed next.
I propose that we consider cell reconditioning as another tool in the arsenal of regenerative strategies. Reconditioning cues frequently lead to cell reprogramming. One recent example of reconditioning of cell environment to achieve reprogramming is provided by the finding that in vivo blockade of CXCL12 in mice with adriamycin-induced nephropathy salvages renal function by inhibiting Notch signaling in podocyte progenitors.21 On the other hand, reconditioning of a particular structural component of the existing damaged cellular pool is a relatively novel aspect of regenerative medicine (Figure 2), which is formed on the (1) solid knowledge of the developing defect of an organelle in a particular disease, (2) appreciation of the pathogenic consequences of such a defect, and (3) design of a therapeutic intervention aimed at restoring the defective organelle. In the past, this term was used exclusively in relation to sport medicine, space flights, and, more recently, in epigenetic reconditioning of tumor cells and in transplantation medicine to improve functions of suboptimal grafts of lung, kidney, and liver.22,23 Occasionally, a specific damaged portion of the cell or a mutant gene/protein can be identified as a hierarchical epicenter of pathogenic pathways that has profound and far-reaching consequences, akin to the opening of Pandora’s box. Cell reconditioning can be achieved by activation of sirtuins or endothelial nitric oxide synthase, both advocated as powerful rejuvenation strategies. A number of small molecule SIRT1 activators have been synthesized. Three generations of sirtuin-activating compounds include, in addition to resveratrol, quercetin, and butein (first generation), SRT1720, SRT1460, and SRT2183 (second generation), and sirtuin-activating compounds 5, 9, and 10 (third generation), all extending life- and health-span. These compounds, presently undergoing clinical trials, represent novel venues of rejuvenation therapy. Another promising compound in this category is NAD+, a cofactor necessary for activation of several sirtuins. NAD+ bioavailability is reduced in disease states and aging and its precursor, nicotinamide, is therapeutic in correcting NAD+ deficiency. On the other hand, endothelial nitric oxide synthase enhancer AVE9488 offers restoration of nitric oxide bioavailability. Examples of cell reprogramming upon reconditioning cell-matrix adhesion are too numerous to recount here.
As an example of organellar diseases, mitochondriopathy of Leigh syndrome could be corrected using combined mRNA-microRNA nonviral approach to repress mutant T8993G mitochondrial DNA.24 In the same vein, mitochondrial-derived peptides humanin and mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) are used to improve insulin sensitivity in mouse models of type 2 diabetes and block cardiac fibrosis in aging mice by virtue of their combined cytoprotective and senolytic effects.
This is also the case with glycocalyx in sepsis, acute ischemia, and diabetes, to name a few. This outermost carbohydrate-rich coat of cells is essential for regulating leukocyte adhesion, permeability, and mechanotransduction, and serves as a repository of survival factors, antioxidants, and other proteins. It is invariably degraded either acutely, as in septic or ischemic injury, or chronically, as in diabetes mellitus, propelling structural and functional deterioration. Through restoration of glycocalyx it should be possible to curtail the entire pathogenic chain of events. We termed such therapeutic intervention reconditioning. In the case of a loss of endothelial glycocalyx, reconditioning translates into detection of (1) degradation of endothelial glycocalyx early in the course of, for instance, septic or ischemic insult; (2) the consequent impairment of microcirculation with propensity toward thrombogenesis, leukocyte egress, increased vascular permeability, and impaired vasomotion, all further aggravating the original insult; and (3) design of a therapeutic strategy to expeditiously restore damaged glycocalyx and, by doing so, revert pathogenetic course of a disease and facilitate innate regeneration. Our recent attempts to recondition endothelial cells in septic mice with liposomal nanocarriers of preassembled glycocalyx25 provide an example of this therapeutic approach. Obviously, such a reconditioning therapy, if successful, could bypass or supplement other, more complicated approaches.
In conclusion, these newer additions to the armamentarium of regenerative medicine, which counter the immutability of cell/organ death, as Mark Twain did with his obituary, are driven by attempts to exploit the potential reversibility of sustained structural and functional damage. In this vein, both cell reprogramming and reconditioning strategies may offer considerable benefits in the future.
Studies from the author’s laboratory are supported in part by National Institutes of Health grant HL144528 and research and education funds from the New York Community Trust.
I would like to extend my apologies to authors of many relevant studies that have not been referenced here because of space limitations.
1. Trounson A, McDonald C: Stem cell therapies in clinical trials: Progress and challenges. Cell Stem Cell 17: 11–22, 201526140604
2. Gurdon JB: From nuclear transfer to nuclear reprogramming: The reversal of cell differentiation. Annu Rev Cell Dev Biol 22: 1–22, 200616704337
3. Srivastava D, DeWitt N: In vivo cellular reprogramming: The next generation. Cell 166: 1386–1396, 201627610565
4. Naiman N, Fujioka K, Fujino M, Valerius MT, Potter SS, McMahon AP, et al.: Repression of interstitial identity in nephron progenitor cells by Pax2 establishes the nephron-interstitium boundary during kidney development. Dev Cell 41: 349–365.e3, 201728535371
5. Kaminski MM, Tosic J, Kresbach C, Engel H, Klockenbusch J, Müller AL, et al.: Direct reprogramming of fibroblasts into renal tubular epithelial cells by defined transcription factors. Nat Cell Biol 18: 1269–1280, 201627820600
6. Watanabe K, Liu Y, Noguchi S, Murray M, Chang JC, Kishima M, et al.: OVOL2 induces mesenchymal-to-epithelial transition in fibroblasts and enhances cell-state reprogramming towards epithelial lineages. Sci Rep 9: 6490, 201931019211
7. Rinkevich Y, Montoro DT, Contreras-Trujillo H, Harari-Steinberg O, Newman AM, Tsai JM, et al.: In vivo clonal analysis reveals lineage-restricted progenitor characteristics in mammalian kidney development, maintenance, and regeneration. Cell Reports 7: 1270–1283, 201424835991
8. Bi B, Schmitt R, Israilova M, Nishio H, Cantley LG: Stromal cells protect against acute tubular injury via an endocrine effect. J Am Soc Nephrol 18: 2486–2496, 200717656474
9. Papadimou E, Morigi M, Iatropoulos P, Xinaris C, Tomasoni S, Benedetti V, et al.: Direct reprogramming of human bone marrow stromal cells into functional renal cells using cell-free extracts. Stem Cell Reports 4: 685–698, 201525754206
10. Matsumoto K, Xavier S, Chen J, Kida Y, Lipphardt M, Ikeda R, et al.: Instructive role of the microenvironment in preventing renal fibrosis. Stem Cells Transl Med 6: 992–1005, 201728297566
11. Ubil E, Duan J, Pillai IC, Rosa-Garrido M, Wu Y, Bargiacchi F, et al.: Mesenchymal-endothelial transition contributes to cardiac neovascularization. Nature 514: 585–590, 201425317562
12. Yang R, Xu X, Li H, Chen J, Xiang X, Dong Z, et al.: p53 induces miR199a-3p to suppress SOCS7 for STAT3 activation and renal fibrosis in UUO. Sci Rep 7: 43409, 201728240316
13. Halloran PF, Melk A: Renal senescence, cellular senescence, and their relevance to nephrology and transplantation. Adv Nephrol Necker Hosp 31: 273–283, 200111692465
14. Yang HC, Rossini M, Ma LJ, Zuo Y, Ma J, Fogo AB: Cells derived from young bone marrow alleviate renal aging. J Am Soc Nephrol 22: 2028–2036, 201121965376
15. Cippà PE, Sun B, Liu J, Chen L, Naesens M, McMahon AP: Transcriptional trajectories of human kidney injury progression. JCI Insight 3: 123151, 201830429361
16. Chen J, Li H, Addabbo F, Zhang F, Pelger E, Patschan D, et al.: Adoptive transfer of syngeneic bone marrow-derived cells in mice with obesity-induced diabetes: Selenoorganic antioxidant ebselen restores stem cell competence. Am J Pathol 174: 701–711, 200919147816
17. Baker DJ, Wijshake T, Tchkonia T, LeBrasseur NK, Childs BG, van de Sluis B, et al.: Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479: 232–236, 201122048312
18. Sturmlechner I, Durik M, Sieben CJ, Baker DJ, van Deursen JM: Cellular senescence in renal ageing and disease. Nat Rev Nephrol 13: 77–89, 201728029153
19. Docherty MH, O’Sullivan ED, Bonventre JV, Ferenbach DA: Cellular senescence in the kidney. J Am Soc Nephrol 30: 726–736, 201931000567
20. Lipphardt M, Song JW, Matsumoto K, Dadafarin S, Dihazi H, Müller G, et al.: The third path of tubulointerstitial fibrosis: Aberrant endothelial secretome. Kidney Int 92: 558–568, 201728476555
21. Romoli S, Angelotti ML, Antonelli G, Kumar Vr S, Mulay SR, Desai J, et al.: CXCL12 blockade preferentially regenerates lost podocytes in cortical nephrons by targeting an intrinsic podocyte-progenitor feedback mechanism. Kidney Int 94: 1111–1126, 201830385042
22. Rosso L, Zanella A, Righi I, Barilani M, Lazzari L, Scotti E, et al.: Lung transplantation, ex-vivo reconditioning and regeneration: State of the art and perspectives. J Thorac Dis 10[Suppl 20]: S2423–S2430, 201830123580
23. Gailhouste L, Liew LC, Yasukawa K, Hatada I, Tanaka Y, Nakagama H, et al.: Differentiation therapy by epigenetic reconditioning exerts antitumor effects on liver cancer cells. Mol Ther 26: 1840–1854, 201829759938
24. Grace HE, Galdun P 3rd, Lesnefsky EJ, West FD, Iyer S: mRNA reprogramming of T8993G Leigh’s syndrome fibroblast cells to create induced pluripotent stem cell models for mitochondrial disorders. Stem Cells Dev 28: 846–859, 2019
25. Zhang X, Sun D, Song JW, Zullo J, Lipphardt M, Coneh-Gould L, et al.: Endothelial cell dysfunction and glycocalyx - a vicious circle. Matrix Biol 71–72: 421–431, 201829408548