The likely role of proteolytic enzymes in unwanted differentiation of stem cells in culture (original) (raw)
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Isolation and differentiation of mouse embryonic stem cells
Iranian Journal of Reproductive Medicine
Background: Recently, embryonic stem (ES) cells have become very important resources in basic medical researches. These cells can differetiate into derivatives of all primary germ layers. Objectives: In order to isolate embryonic stem cells in vitro, the blastocyst were cultured and the morphological aspects, population doubling time, alkalin phosphatse and differentiation properties of the cells were investigated. Materials and Methods: The balstocysts from NMRI mice were cultured for 3 days up to time that inner cell mass (ICM) reach to the outgrowth stage. The cells were disaggregated and trypsinized every 3 days until the appearance of the colonies of ES cells. The colony positive cells were fixed and stained for alkaline phosphatase. The ES cells were cultured in suspension state for 5 days, at the same time Leukaemia Inhibitory Factor (LIF) was removed from media to form embryoid bodies(EBs). The EBs were cultured for 8 -20 days on collagen coated dish to induce the spontaneouse differentiation. Results: During the 6-9 days after the disaggregation of ICM in the expansion stage , the colony of ES cells appeared as a flat monolayer mass with strike boundaries and nondistinguish cytoplasm including a few nuclei. In colony formation stage, the morphology changed from flat monolayer to round multilayer with strike define boundaries. Undifferentiated cells were seen as intensely small cells attached together compactly with high nucleus/cytoplasm (N/C) ratio. The cells of colonies tend to differetiate by separation from each other and became larger and diffused on substrate by attaching to dish. The positive alkaline phosphatase cells were seen in typical morphology of ES colonies. The EBs cells were seen in culture after 5 days in suspension and began to spontaneously differentiate into various types of cells such as nerve and hematopoitic lineages. Conclusion: Despite strike morphology of ES colonies, it is difficult to distinguish the differentiated from undifferentiated cell colonies in the colony formation stage. New ES cells are capable to give rise into EBs and are susceptible of spontaneously differentiation in various type of cells.
Genes & Development, 1990
Differentiation inhibiting activity/leukemia inhibitory factor (DIA/LIF) is a glycoprotein that controls differentiation of pluripotential stem cells. Alternative transcription generates both diffusible and matrixassociated forms of DIA/LIF. Transcriptional analysis using a sensitive ribonuclease protection assay revealed that the two messages are expressed independently, consistent with the proposition that the two forms of DIA/ LIP have distinct biological roles. DIA/LIF expression was found to be activated early during differentiation of embryonic stem (ES) cells, providing a mechanism for feedback regulation of stem cell renewal. Expression of DIA/LIF by mesenchymal cells was shown to be controlled in a paracrine manner by polypeptide regulatory factors. Specific expression of the two forms of DIA/LIF was also demonstrated in the egg cylinder-stage mouse embryo. The combination of cell type-specific and signal-specific regulation enables very precise control over DIA/LIF expression and may represent an important component of the regulatory networks that govern stem cell proliferation and differentiation during mammalian development.
Elimination of damaged proteins during differentiation of embryonic stem cells
Proceedings of the National Academy of Sciences of the United States of America, 2006
During mammalian aging, cellular proteins become increasingly damaged: for example, by carbonylation and formation of advanced glycation end products (AGEs). The means to ensure that offspring are born without such damage are unknown. Unexpectedly, we found that undifferentiated mouse ES cells contain high levels of both carbonyls and AGEs. The damaged proteins, identified as chaperones and proteins of the cytoskeleton, are the main targets for protein oxidation in aged tissues. However, the mouse ES cells rid themselves of such damage upon differentiation in vitro. This elimination of damaged proteins coincides with a considerably elevated activity of the 20S proteasome. Moreover, damaged proteins were primarily observed in the inner cell mass of blastocysts, whereas the cells that had embarked on differentiation into the trophectoderm displayed drastically reduced levels of protein damage. Thus, the elimination of protein damage occurs also during normal embryonic development in v...
Concise Review: Fate Determination of Stem Cells by Deubiquitinating Enzymes
Post-translational modification by ubiquitin molecules is a key regulatory process for stem cell fate determination. Ubiquitination and deubiquitination are the major cellular processes used to balance the protein turnover of several transcription factors that regulate stem cell differentiation. Deubiquitinating enzymes (DUBs), which facilitate the processing of ubiquitin, significantly influence stem cell fate choices. Specifically, DUBs play a critical regulatory role during development by directing the production of new specialized cells. This review focuses on the regulatory role of DUBs in various cellular processes, including stem cell pluripotency and differentiation , adult stem cell signaling, cellular reprogramming, spermatogenesis, and oogenesis. Specifically, the identification of interactions of DUBs with core transcription factors has provided new insight into the role of DUBs in regulating stem cell fate determination. Thus, DUBs have emerged as key pharmacologic targets in the search to develop highly specific agents to treat various illnesses. STEM CELLS 2016; 00:000–000 SIGNIFICANCE STATEMENT The review is written from a unique perspective in overseeing the regulatory role of deubiquiti-nating enzymes in stem cells. A great attention has been given to the physiological role of ubiquitination system in regulating pluripotency of embryonic stem cells. However, the reversal of ubiquitination by deubiquitinating enzymes plays an equally important role in the regulation of levels of expression of the stemness-related proteins by preventing its ubiquitination. This is timely topic, given that this is the first review article attempting to discuss the accumulated evidence suggesting the critical role of deubiquitinating enzymes in regulating stem cell fate determination process such as maintenance of pluripotency, differentiation, cellular reprogramming , spermatogenesis, and oogenesis. Beyond this, the review provide us the clue for the remaining challenges in developing DUBs as targets for stem cell therapeutics.
Proteomic studies of stem cells
stembook.org
Stem cells of both embryonic and adult origins hold great promise in regenerative medicine owing to their unique properties of unlimited self renewal and differentiation toward specific lineage(s) once they receive the proper signals. Proteomics is a series of technology platforms driven by advancements in mass spectrometry and bioinformatics that encompass protein identification, the relative quantitation of proteins and peptides, their subcellular localization, and studies of post-translational modifications and protein-protein interactions. Stem cell biology has been influenced by these approaches and has evolved in the post-genomics era. Among many challenges in stem cell biology, there is a pressing need for the implementation of proteomic applications. Recent work on stem cells using proteomics has shown that transcriptome analyses fail to provide a full guide to developmental change in stem cells, and protein interactions that can only be discovered systematically using proteomic approaches have yielded important new concepts on processes regulating development and stem cell pluripotency. In this chapter, we will review current proteomic studies on embryonic and adult stem cells with an emphasis on embryonic stem cells.
Cysteine Proteases in Differentiation of Embryonic Stem Cells into Neural Cells
Stem Cells and Development, 2011
Glycosylated mouse cystatin C (mCysC), an endogenous inhibitor of cysteine cathepsin proteases (CP), has been suggested as a cofactor of b-FGF to induce the differentiation of mouse embryonic stem cells into neural progenitor cells (NPCs). To investigate the possible role of CP in neural differentiation, we treated embryoid bodies (EBs) with (i) E64, an inhibitor of papain-like CP and of calpains, (ii) an inhibitor of cathepsin L (iCatL), (iii) an inhibitor of calpains (iCalp), or (iv) cystatins, and their ability to differentiate into neural cells was assessed. We show that the inhibition of CP induces a significant increase in Pax6 expression in EBs, leading to an increase in the number of nestin-positive cells after 3 days. Fourteen days after E64 treatment, we observed increased numbers of b-III-tubulin-positive cells, showing greater percentage of immature neurons, and this feature persisted up to 24 days. At this point, we encountered higher numbers of neurons with inward Na + current compared with untreated EBs. Further, we show that mCysC and iCatL, but not unglycosylated egg white cystatin or iCalp, increased the numbers of NPCs. In contrast to E64 and iCatL, mCysC did not inhibit CP in EBs and its neural-inducing activity required b-FGF. We propose that the inhibition of CP induces the differentiation of mouse embryonic stem cells into NPCs and neurons through a mechanism that is distinct from CysC-induced neural differentiation.
The Loss of Phenotypic Traits by Differentiated Cells
Journal of Experimental Medicine, 1969
When mitotically quiescent chick chondroeytes are liberated from their polysaccharide matrix and cultured on a fibrin dot in the presence of embryo extract, the following is observed: (a) Within 18 hr the rounded chondrocytes transform into fibroblastic cells and cease synthesizing chondroitin sulfate. (b) Within 24 hr over 50% of the cells incorporate thymidine, and by 35 hr over 95% have completed at least one cell division. (c) The amount of chondroitin sulfate synthesized per cell in the log phase of growth is less than 5% that of the nondividing progenitor cells. (d) If after 7-10 days such fibroblastic chondrocytes contact their siblings, many resume a rounded shape and synthesize chondroitin sulfate in quantifies characteristic of their in vivo progenitors. (e) When cultured as fibroblastic cells for over 2 wk and then allowed, at high density to contact siblings, they neither round up nor deposit chondroitin sulfate. (/) Fibroblastic chondrocytes obtained by protracted culturing do not display the surface affinities of normal chondrocytes. Chondrocyte progeny which do not deposit chondroitin sulfate or aggregate with one another or with normal chondrocytes have been termed "dedifferentiated" or "altered" chondrocytes (1-5). Many of these observations have been confirmed by Kuroda (6) and Shuiman and Meyer (7) with chick chondrocytes, by Chiakulas (8) with frog chondrocytes and by Manning and Bonher (9) with human chondrocytes. Coon's (10) report on cloned chondrocytes both supports and conflicts with the above analysis. Coon made the important observation that many dedifferentiated chondrocytes which do not synthesize chondroitin sulfate in high density cultures, do yield progeny which synthesize the polysaccharide if cultured at low density. In addition, Coon and Calm (11) and Calm, Coon, and Calm (12) claim that a high molecular weight, heat-labile factor in embryo extract selectively inhibits the synthesis of chondroitin sulfate by chondrocytes and melanin by pigment ceils (see however, 13, 14).
Toward an Understanding of the Physiological Function of Mammalian Stem Cells
Developmental Cell, 2005
stand where and when stem cells are actually present in vivo, what they are actually doing, and how these Howard Hughes Medical Institute and Departments of Internal Medicine and physiological functions are regulated. On the other hand, if the goal is to generate differentiated cells for Cell and Developmental Biology 1500 East Medical Center Drive drug screening, toxicity screening, or even for certain cell therapies, then it may not matter whether the stem University of Michigan Ann Arbor, Michigan 48109 cells that form the differentiated cells have acquired some of their properties in culture. The lines between these divergent aims are not al-Summary ways clear. Recent evidence indicates that it is possible to derive cell lines with compelling stem cell properties Stem cell biology has the potential to yield new therafrom the long-term culture of adult tissues that do not pies, new insights into disease, and a clearer undernecessarily exhibit similar properties in vivo (Pittenger standing of tissue formation and maintenance. Howet al., 1999; Jiang et al., 2002; Li et al., 2003; Zhao et ever, much of what we know about many stem cells al., 2003; Kanatsu-Shinohara et al., 2004; Kogler et al., is based upon experiments performed in culture. 2004). Do these cell lines reflect previously unappreci-Stem cells sometimes exhibit critical differences in ated but physiologically important stem cells that are their properties or regulation between the culture and active in vivo, or do they reflect cells that lack stem in vivo environments. Though cell lines with stem cell cell properties in vivo and acquire such properties by properties can be derived from the long-term culture reprogramming in culture? If these cells acquire unof diverse tissues, it is not clear whether cells with physiological properties in culture, will they represent similar properties exist in vivo. If the goal is to use stable sources of differentiated cells that are normal differentiated cells for therapy or drug screening, it enough to be therapeutically useful? may not matter whether these stem cells exist in vivo. To navigate these complex questions, it is important However, to understand tissue development/mainteto clearly recognize what we know and what we don't. nance or the role of stem cells in disease, it is impor-In the words of Yogi Berra, "You can observe a lot just tant to characterize progenitor function in vivo to by watching" but "You've got to be very careful if you evaluate physiological significance. don't know where you're going, because you might not get there." Yogi's meaning shines through the internal Stem cells are capable of self-renewal and the generaambiguities and inconsistencies in his words, just as tion of large numbers of differentiated progeny, often the promise of stem cell research is not dulled by its including multiple types of progeny. Beyond these criinherent ambiguities and inconsistencies. Nonetheless, teria, stem cells distinguish themselves from other prowe are much more likely to get where we want to go if genitors by having the broadest developmental potenwe are thoughtful about potential disconnects between tial in a particular tissue at a particular time. Different physiological function and therapeutic utility. types of stem cells have different developmental potentials, including pluripotent embryonic stem cells that The Disconnect between Developmental Potential can generate every cell type in the body and multipoin Culture and In Vivo tent stem cells, such as hematopoietic stem cells, that There are several well-documented examples of progenerate diverse cells in a particular tissue but gengenitors that broaden their developmental potential in erally lack the ability to make cells in other tissues. culture in ways that do not appear to occur under phys-Some stem cells, like hematopoietic stem cells, have iological circumstances (Figure 1). During normal develbeen extensively studied in vivo, and their physiological opment, primordial germ cells give rise primarily or exfunction is well characterized. Other stem cells, like clusively to germ cells and their progenitors (Wei and embryonic stem cells, have been derived in culture and Mahowald, 1994). When primordial germ cells are inhave properties that differ from the blastocyst inner cell jected into blastocysts, they do not detectably contribmass from which they arise (Nichols et al., 2001). Alute to developing embryonic tissues, suggesting that though embryonic stem cells are a culture "artifact" in they are committed under physiological circumstances the sense that they acquire certain properties that are to the germline lineage (Donovan, 1994). However, if not exhibited in a blastocyst, they remain capable of these cells are cultured for 6 days in the presence of generating functionally normal cells in all tissues (Nagy steel factor, fibroblast growth factor, and leukemia inet al., 1993). hibitory factor, they form colonies with characteristics The fact that stem cells sometimes acquire unphysioof embryonic stem cells and early passage lines delogical but potentially therapeutically useful properties rived from these colonies contribute to all tissues upon in culture creates a tension. On the one hand, we study injection into blastocysts (Matsui et al., 1992; Donovan, stem cells partly to understand their role in develop-1994). This demonstrates that primordial germ cells can ment and disease. For these aims, it is critical to underbe quickly reprogrammed in culture to exhibit a broader developmental potential. Postnatal somatic progenitors can also be repro