Stem Cells and Aging

The purpose of the present article, presented at the 4th Conference of ESAAM, which took place on October 30–31, 2015, in Athens, Greece, was not to cover all aspects of cellular aging but to share with the audience of the Conference, in a 15-minute presentation, current knowledge about rejuvenating and repairing somatic stem cells that are distinct from other stem cell types (such as embryonic stem cells [ESCs] or induced pluripotent stem cells [iPSCs]), emphasize that our body in old age cannot take advantage of these rejuvenating cells, and provide some examples of novel experimental stem cell applications in the field of rejuvenation and antiaging biomedical research.

Stem cells are cells that not only multiply themselves by cell division (mitosis) but also differentiate into one or more types of other cells, which are completely different phenotypically from the mother stem cell. There are distinct categories of stem cells. ESCs that originate from the inner cell mass of the blastocyst and can form embryonic bodies, differentiating into any other useful cell of the organism during embryonic development and growth. iPSCs that have similar properties with the ESCs and can be produced from any adult cell type by inserting into their genetic material four transcription factors (Oct4, Sox2, cMyc, and Klf4). ¹ Finally, adult stem cells (ASC) that are found in the body after birth and support regeneration and wound healing of the organism during life. ASCs can be monopotent, oligopotent, or multipotent. Only ESCs and iPSCs, which are embryonic-like stem cells, are totipotent and can form embryonic bodies. ²

ESCs and iPSCs can be expanded to unlimited growth without Hayflick limit and differentiated in vitro. ESC and iPSC can be differentiated into any other useful cell of the body, providing thus a precious material for the rejuvenation of the organism. Furthermore, iPSCs, developed from the patient's own cells, are autologous and do not face rejection by the immune system of the host. ESCs were first isolated from mouse blastocysts and cultured in 1981 independently by two laboratories, in England ³ and in the United States. ⁴ Only after 17 years, in 1998, the first human origin ESC line was established. ⁵ However, cells derived by ESCs, not being autologous, were a subject of rejection by the immune system if used for the replacement of mature cells upon differentiation, when transplanted to the patient. A solution for this problem would be the creation of autologous ESCs from the patient's own cells. Cloning, the production of blastocysts using an animal's adult cells, was known to be possible for frogs since 1962, ⁶ and at 1997, the birth of first mammalian clone, the sheep Dolly, was announced. ⁷ The creation of autologous stem cells by human cloning (therapeutic cloning) was possible, however, not desired due to ethical considerations. This problem was solved at 2007 by the creation of ESC lines bypassing cloning using the technique of iPSCs. ¹ Using this technique, medicine could produce autologous differentiated cells for the replacement of dead or damaged cells in the body. Also, next year, in 2008, Stemagen, a California, USA, based biotechnology company announced the creation of the first human origin iPSC line, the first “human clone.” ⁸ The possibility to create autologous ESCs such as iPSCs was considered a very significant breakthrough that opened new ways in medicine, and accordingly, Sir John B. Gurdon who first achieved cloning, together with Shinya Yamanaka who invented the technique for the creation of iPSCs, was awarded the Nobel Prize for physiology and medicine in 2012.

A considerable reduction of reprogramming efficiency of iPSC has been observed with increased donor age, ^9,10 and as a consequence, these autologous cells could not be easily created at old age, when needed the most. On the contrary, theoretically, only one single colony might be sufficient for future applications. Deletion of the anti-oncogene p53-p21 has been proposed to increase the efficiency of iPSC generation. ¹¹ Alternatively, iPSCs can be generated from young cells collected on birth with only two gene modifications, the insertion of oct4 and sox2 genes in their genome ¹² ; thus, preserving young cells or generating iPSCs while young may be beneficial for future old age patients. There is not yet any approved product based on ESCs or iPSCs. There are only four clinical trials announced, the first conducted by the company Geron has been interrupted for safety reason. It is widely known that these cells usually induce the development of tumors, mostly teratomas.

On the contrary, a different kind of stem cell has been discovered. In 2002, the group of Catherine Verfeillie's, working at the University of Minnesota, discovered that a subpopulation of mesenchymal stem cells (MSCs) existing in the bone marrow are multipotent stem cells that can differentiate into several other cell types. ¹³ These cells have been named multipotent adult progenitor cells (MAPC) ¹⁴ and initially raised a lot of controversy. Following the first publication, the above results have been initially doubted by other groups, who reported inability to duplicate similar data and the research team was accused for scientific misconduct. ¹⁵ However, the results of this remarkable article have been confirmed and corroborated multiple times still to date by many other researchers internationally. Today, the original article published in Nature is cited more than 6000 times (Google scholar search). Numerous research teams worldwide not only replicated and verified the existence of multipotent cells in the bone marrow cells but also used these for experimental therapeutic applications. ^16,17 A copy of Catherine Verfeillie's notebook page marking her discovery has been grafted on the wall of discoveries of the University of Minnesota campus as one of the biggest discoveries made in this institution.

More recently, in a similar pattern, considering another type of somatic stem cell (very small embryonic-like stem cells [VSEL]), a Japanese research group was not able to confirm the findings of Kucia et al. ¹⁸ using adult mice ¹⁹ ; however, several other groups worldwide were able not only to isolate and characterize but also use these cells for experimental therapies. ^{20 –23}

Multipotent stem cells have been described in various tissues of the postnatal body with different names such as marrow-isolated adult multilineage inducible cells (MIAMI cells), ²⁴ unrestricted stem cells in umbilical cord blood, ²⁵ VSEL in bone marrow, ^18,26 hemangioblasts in bone marrow, ²⁷ endothelial precursor cells (EPC), multipotent ASCs in the dermis, ²⁸ multilineage differentiating stress enduring (Muse) cells, a type of MSCs, ²⁹ and spore-like cells (similar to VSEL, but described since 2001). ³⁰ Multipotent cells, collectively named ASCs, have been found not only in bone marrow and cord blood but also in adipose tissue (adipose-derived stem cells [ADSCs]) ^31,32 and many other tissues. ³³ In addition, a type of stem cell supporting cells, telocytes, was found in the cardiac and other tissues. ^{34 –36} Somatic ASCs are multipotent and do not form teratomas, and therefore, these cells have been safely used in clinical trials.

Soon some species of ASCs, mainly MSCs, such as MAPCs and ADSCs, as well as ASC containing mixed cell populations, such as bone marrow and adipose tissue-derived vascular fraction, have been tested for autologous and allogeneic therapies in experimental animals and clinical trials and, in contrast to ESCs and iPSCs, without any complications. ^17,37 These cells are multipotent having the capacity of multilineage differentiation without crossing the embryonic layer limits. ^16,17,38 Their final differentiation in the target tissue of repair may be guided by external stimuli, the local extracellular matrix and cell environment. ³⁸ However, Lengner et al. were not able to show that inducible ablation of Oct4 expressing cells in the adult mouse had effect in tissue homeostasis. ³⁹

A hierarchy in the development of these cells has been suggested with MIAMI cells being the mother cells of MSCs, EPCs, and hematopoietic stem cells, ³⁷ although the same role has been also assigned for hemangioblasts. ⁴⁰ The possible origin of somatic adult multipotent cells has been recently reviewed. ⁴¹

Several cell types such as nerve stem cells and neurons can be produced in vitro from ESCs or iPSCs or by stem cells collected after birth such as umbilical cord blood stem cells. ^42,43 MSCs in vivo can home to the site of damaged tissues, promote tissue regeneration, and decrease inflammation providing thus a useful tool for therapeutic applications. ⁴⁴ Numerous preclinical studies have shown the therapeutic potential of these cells, for example, Honma et al. reported that immortalized MSCs can differentiate in vivo into neurons, protecting against injury in a rat experimental ischemia model. ⁴⁵ There are five FDA-approved preparations and one European Union-approved preparation (Holoclar) that contain ASC. As indicated by a monitoring Swiss group, the therapeutic applications of ASC are constantly increasing in the last years in Europe. ^{46 –49} Today, hundreds of clinical trials are open worldwide evaluating the safety and clinical utility of ASCs. ⁵⁰ Autologous ASCs collected from the patient, either from bone marrow or from the adipose tissue, are used for most of these therapies and clinical trials. However, the efficacy of these therapies is not sufficient at the time needed most, at old age. Stem cells found in human body after birth are a subject of senescence and therefore lose their capacity to multiply, to differentiate, and to support tissue regeneration with the progress of age because of senescence. In addition, an increased susceptibility to stem cell death on stress has also been reported. ^51,52

This cellular senescence seems to contribute dramatically not only in the senescence of the organism but also in the development and progress of diseases of old age such as atherosclerosis, type II diabetes arthritis, and cancer, ^{53 –55} and therefore, as Sharpless and Depinho remarked, “we are aging because the rejuvenating stem cells are aging.” ⁵³

Accordingly, it has been reported that hematopoietic and multipotent stem cells derived from old adults have different functions and surface protein markers, diminished translating activity, and suffer greater loss of integrity of their genome, compared with cells derived from younger donors. ⁵⁶ Wagner et al. reported 67 age-induced and 60 age-repressed genes in MSCs and 432 age-induced and 495 age-repressed genes in hematopoietic stem cells. ⁵⁷ Age-associated loss of mitochondrial genome integrity of stem cells has been also reported. ^51,52 It is also evident that bone marrow stem cells originating from young donors are more effective for cardiovascular therapies in comparison to cells originating from aged donors. ⁵⁸ In preclinical experiments, infusions of bone marrow and MSCs derived from young mice into aged mice resulted in a delay of aging, postponed reproductive failure, and improved the survival of offspring. ^59,60 Furthermore, Jaskelioff et al. reported a reversal of aging in telomerase-deficient mice, after telomerase reactivation, attributing this effect to ASC reactivation. ⁶¹ On the cellular level, reversal of stem cell aging has been reported by suppressing mitochondrial stress ^51,52,62 and stem cell intrinsic reduction of Wnt5a expression. ⁶³

Transplanting stem cells derived from a young pig into an aged pig's skin resulted in skin rejuvenation. ⁶⁴ In contrast, it has been reported that epidermal stem cells may escape the effects of aging, considering gene function and DNA damage. ⁶⁵ However, epidermal stem cell function is inhibited by age-associated inflammation. ⁶⁶ ADSCs have been reported to reduce inflammation. ⁴⁴ Accordingly, after subcutaneous injection in hairless mice, these cells have been shown to increase collagen synthesis, dermal thickness, collagen density, fibroblast number, and angiogenesis. ^67,68 Similar results have been recently reported in a clinical skin antiaging setting. ⁶⁹ In our experience, these cells after subcutaneous injection are situated around new blood vessels, ⁷⁰ however, other researchers attribute MSC skin antiaging effect into epithelial transdifferentiation. ⁷¹

Although a lot of money and effort have been invested on ESC and iPSC research, today, the vast majority of stem cell clinical trials and experimental cell therapies are based on somatic ASCs. However, these cells age, and there is considerable evidence that “we age because our stem cells age.” ⁵³ Techniques that will allow the reversal or avoidance of somatic stem cell aging or their replacement with younger cells may in the future allow the extension of the healthy part of human life.