○Marrow-isolated adult multilineage-inducible cells derived from the bone marrow of vertebral bodies under low oxygen conditions were reported to be particularly efficient in differentiating into neural lineages
○Very small embryonic-like cells (VSELs)
○MSCs derived from bone marrow as well as other sources (excluding adipose tissue)
○SSEA-positive cells with enhanced expansive and clonogenic potential
”謎めいた細胞”は、細胞にとって過酷な条件の元で生まれる。 こうした胚様の多能性を持つ細胞が生じる現象は、いまだ未解明である。 （However, the diversity of reports on enigmatic cells with morphology similar to embryonic counterparts that were isolated under harsh conditions may signal that this is an unelucidated phenomena.）
以下は、その一部です。うんちくのある文章です、 Starting in the early 2000s, several reports about spontaneously occurring pluripotent cell types emerged. Derived from mice and human bone marrow by negative depletion of CD45 (+)/glycophorin (+) cells, multipotent adult progenitor cells (MAPCs) were reported to undergo triploblastic differentiation under defined conditions in vitro. MAPCs did not form teratomas, contributed to chimaera formation when injected into mouse blastocysts, and contributed to cardiac regeneration in severe combined immune-deficient (SCID) mice[19,20]. Marrow-isolated adult multilineage-inducible cells derived from the bone marrow of vertebral bodies under low oxygen conditions were reported to be particularly efficient in differentiating into neural lineages without displaying features of pluripotency. Very small embryonic-like cells (VSELs) were isolated from murine bone marrow by positive selection for the chemokine receptor CXCR4 and were shown to display features of embryonic cells (cell and nuclei size, chromatin characteristics, telomerase activity). The authors hypothesized that such cells with embryonic-like surface markers [stage-specific embryonic antigen (SSEA), OCT-4, and NANOG] could be epiblast-derived pluripotent cell remnants of embryonic developmental stages; these cells could be a less controversial source for regenerative approaches. The existence of VSELs was challenged a couple of year later as other groups failed to replicate their isolation from bone marrow Remarkably, almost all reports of bone marrow-derived cells that claimed to retain embryonic-like stem cell features were isolated in modified culture conditions (such as low oxygen tension or serum deprivation). Arguments that such cells are early MSC progenitors or culture condition-modified MSCs have not been fully investigated to date. MSCs derived from bone marrow as well as other sources (excluding adipose tissue) were shown to foster a population of SSEA-positive cells with enhanced expansive and clonogenic potential. Arguments that SSEA-positive cells are a culture artefact have not been addressed yet. The existence of adult pluripotent cell populations proved hard to replicate, leading to doubt concerning the accuracy of the reported findings and concept of naturally occurring pluripotency. However, the diversity of reports on enigmatic cells with morphology similar to embryonic counterparts that were isolated under harsh conditions may signal that this is an unelucidated phenomena.
Adipose-derived pluripotent cells
Isolated from adipose-derived stromal vascular fraction, adipose-derived MSCs (ADSCs) were shown to differentiate to non-mesodermal lineages under special culture conditions in vitro. Notably, the majority of non-mesenchymal lineage differentiation protocols involve an intermediary step including suspension culture, spheroid formation of intermediary progenitors and sometimes serum deprivation. Undifferentiated or in vitro pre-differentiated ADSCs were shown in several reports to contribute to liver, Schwann cell and glial cell regeneration. The advent of IPSCs and the enthusiasm for their potential in generating patient-specific pluripotent cells for research and therapy seemed to throw the controversy of adult pluripotency into oblivion. However, two special cell types continue to capture research interest: multilineage differentiating stress-enduring cells (MUSE) and dedifferentiated fat cells.
MUSE cells were initially identified by applying stressful culture conditions to several cell populations such as MSCs[27,28]; they have been further obtained from adipose tissue by positive immune-separation for the mesenchymal marker CD105 and SSEA-3. MUSE cells are capable of triploblastic differentiation without tumour formation after in vivo injection into SCID mice; these were considered safer sources for pluripotent cells than ESCs or IPSCs. With several distinctive properties in vitro and in vivo, MUSE cells display low telomerase activity and a normal karyotype. MUSE cells form distinctive clusters in vivo (the so-called M clusters) that resemble ES or IPSCs behaviours in similar conditions. These cells express pluripotent markers, such as NANOG, Oct3/4, Par-4, and Sox2, and are capable of spontaneous or induced expression of mesodermal, endodermal or ectodermal markers. The low levels of cell proliferation and oncogenesis gene expression might account for their low proliferation and absence of tumourigenic activity, while the expression of gene clusters related to death and survival that are shared with non-mammalian species might represent a highly-conserved mechanism of cell survival during extreme conditions. Several preclinical studies have reported their migratory potential due to expression of chemokines involved in cell homing and their capability to participate in liver, kidney, and neural regeneration in relevant animal models (for review see 30). Muse cells also have immunomodulatory properties in lipopolysaccharide-stimulated macrophages and antigen-challenged T-cell assays through downregulating the secretion of pro-inflammatory cytokines (interferon-γ and tumour necrosis factor-α); this effect is probably acquired by transforming growth factor-β1 expression that decreases the immune-regulatory activity through T-box transcription factors in T cells. Interestingly, MUSE cells have been identified in very low numbers in the blood stream of early stage patients with acute stroke where they probably mobilized from bone marrow; MUSE cells have also been detected in situ and in post-mortem bone marrow samples harvested from subjects with severe conditions such as stroke and myocardial infarction. Research to harness the therapeutic potential of such cells for regenerative applications is ongoing; however, their anatomical location in niches has not yet been identified. It is unclear whether induced or naturally occurring stressful conditions are sorting or generating MUSE cells through adaptative and potentially “reprogramming” mechanisms attempting regeneration after major insults.
Adipose tissue was one of the first sources reported for generating MUSE cells and another reportedly pluripotent adult human cell source is dedifferentiated adipose-derived cells (DFATs). Mature adipocytes isolated from adult human adipose tissue that are subjected to an in vitro dedifferentiation strategy (ceiling culture) revert to a more primitive phenotype and gain proliferative and differentiative abilities. Indeed, DFATs were found to have triploblastic differentiation potential in vitro and do not generate teratomas when injected in immuno-deficient mice. As opposed to ADSCs that are obtained by enzymatic digestion of adipose tissue and selection of plastic-adherent fibroblastoid elements, DFATs are homogenous populations. DFATS display surface markers for CD13, CD29, CD44, CD90, CD105, CD9, CD166 and CD54, and do not express CD14, CD31, CD34, CD45, CD66b, CD106, CD117, CD133, CD146, CD271, CD309, HLA-DR and alpha-smooth muscle cell actin; a fraction of DFATs also express SSEA-3. Inter-donor and interspecies variability in the makeup of surface antigens has been reported. Combined with a Poly-D, L-lactic-co-glycolic acid scaffold, rat DFAT cells were able to regenerate periodontal tissue, opening exciting avenues for oral and maxillofacial tissue regeneration[39,40].
Dedifferentiation as a source of adult pluripotent cells
Mature adipocytes are not the only cells capable of dedifferentiation. Mature chondrocytes isolated from the well organized and highly structured cartilage ECM dedifferentiate while in monolayer culture. When expanded in MSC growth medium with or without fibroblast growth factor (FGF), costal chondrocytes express features of MSCs but retain their chondrogenic potential when injected in vivo for cartilage defects. Cartilage progenitor cells with clonogenic and migratory potential reside in osteoarthritic cartilage but not in normal mature cartilage. However, further reports identified surprisingly high levels of the stem cells markers Notch-1, Stro-1 and VCAM-1 in normal cartilage and in a stage- and zone-dependent manner in osteoarthritic (OA) cartilage. Despite their controversial nature, these studies revealed the previously ignored dynamic activity of adult cellular cartilage elements that could be metabolic- and/or mechano-stimulation-dependent. Hypothetically, cells with surface markers of pluripotency in adult cartilage could originate from dedifferentiated chondrocytes induced by metabolic and/or mechanical stress. Disturbances in these parameters might lead to abnormal cell clustering and ECM disorganization that synergizes to produce the progressive cartilage breakdown of OA. The fibrous remodelling of joint surfaces seen in advanced OA stages might represent an abnormal differentiation of such dedifferentiated adult chondrocytes.
Other cell types were shown to successfully dedifferentiate in vitro into multipotent or pluripotent progenitors. Adult human thyrocytes regained multipotency, proliferated and differentiated to neurogenic and adipogenic lineages in vitro. Terminally differentiated keratinocytes were converted to their progenitor cells under FGF induction, while pancreatic islet cells morphed into duct-like progenitor cells under epidermal growth factor exposure. It is noteworthy to mention that these reports involve in vitro cell populations. Isolation protocols require breakdown of ECM structures, a process commonly achieved by maintaining cells in monolayer cultures. Intriguingly, reports about in vivo formation of DFAT cells after induced local mechanical stress in mice might suggest that this process occurs as a natural adaptative mechanism to local stressful conditions. Dedifferentiation, a common mechanism in plants and a limited number of vertebrates that is used for regeneration, involves switching off genes responsible for cell-specific functions, re-entering the cell cycle and proliferating, and switching on “pluripotency”-related genes. This might be a conserved phenomenon in mammalian organisms including humans. Several factors such as hypoxia, prolonged stress and injury are known to induce dedifferentiated cells after in vitro manipulation or in vivo. Factors that naturally induce such phenomena in vivo and the fate of the regenerative processes they launch need further investigation. Reports about dedifferentiation processes occurring in human malignant tumours, such as liposarcomas dedifferentiating to osteosarcomatous components or soft tissue sarcomas to liposarcomas, reflect several rare situations of pathological dedifferentiation processes. Physiological lung myofibroblast dedifferentiation after tissue injury and inflammation accounts for adaptative apoptosis and bronchiolar re-epithelialization. During ageing, impaired dedifferentiation accounts for continued myofibroblast accumulation, excessive matrix deposition and subsequent interstitial lung fibrosis.
In early 2014, a paper described a “unique cellular reprogramming phenomenon” of exposing adult differentiated cells to low pH. CD45-positive spleen lymphocytes from 1-week-old C57BL/6 mice carrying an Oct4-gfp transgene and adult cells derived from the brain, skin, muscle, fat, bone marrow, lung and liver that were transiently exposed to low pH were reported to acquire pluripotency in vitro. A portion of such cells, which the authors termed stimulus-triggered acquisition of pluripotency (STAP) stem cells, were shown to express pluripotency markers, differentiate to triploblastic lineages under specified conditions, and contribute to chimaeras and germline transmission when injected into mouse blastocysts. Compared to mouse ESCs, STAP cells displayed limited self-renewal capability in ES-specific media and did not form colonies in dissociated culture. The authors hypostatised that “unknown cellular mechanisms” triggered by sublethal stress unlocked the cells from their differentiated state and allowed re-expression of pluripotency-related genes, reflecting early embryonic stages. Such phenomena do not likely occur in vivo-at least not in mammalian organisms-as presumed mechanisms block progression from the initial OCT-4 activation to further reprogramming. Several months later, the paper was retracted due to “errors classified as misconduct” by the institutional investigation committee. The negative impact of the retraction was further combined with news about possible “honour suicide” of one of the senior authors. However, while the “multiple errors” could indeed impact the study reproducibility and the credibility of the reported data, they could not rule out the existence of the STAP phenomenon. Interestingly, a recent paper reported a method of preconditioning adult human umbilical cord blood-derived stem cells to increase survival after transplantation. Exposure to oxidative stress and serum deprivation increased cell resistance in vitro, possible pointing to an adaptative mechanism for cell survival.
おまけ 上記論文は、いくつか、興味深い論文を紹介してます。 44. Suzuki K, Mitsutake N, Saenko V, Suzuki M, Matsuse M, Ohtsuru A, Kumagai A, Uga T, Yano H, Nagayama Y, Yamashita S. Dedifferentiation of human primary thyrocytes into multilineage progenitor cells without gene introduction. PLoS One. 2011;6:e19354. PMID: 21556376