Essay: Adaptation in cells

One central role of evolutionary biology is understanding the process of adaptation (Orozco-Terwengel et al., 2012). Culture adaptation in cells describes the phenomenon of occurring karyotypic changes in cell lines after transferring them out of their natural environment (in vivo) into a new environment, the growth medium (in vitro), where the cells are exposed to unaccustomed living conditions. The goal of isolating cells from their living tissues, is to establish a culture for keeping them in continuous state for many years, thus creating unlimited supply and bypassing ethical concerns. Sub culturing or passaging of cells is used to prolong their life and/or expand the number of microorganisms. The isolation of cells from living tissues allows to grow them under controlled conditions and creating cell lines developed from a single cell (Hudu et al., 2016; Kaur and Dufour, 2012).
Stem cells for example, owing the ability of perpetual self-renewal and differentiation into all different cell types of the body (Biehl and Russell, 2009; Rebuzzini et al., 2015), allow the production of organoids and tissues, that can be used drug screening. Additionally, if paired with other approaches like gene editing, SC have the potential to revolutionise cell biology (Drubin and Hyman, 2017; Madl et al., 2018). However, cultivation of human stem cells (hSC), which should maintain the homologous state of the derived cell lines, showed the introduction of chromosomal aberrations after long-term passaging, which raises major concerns about their use in clinical approaches, especially in the field of regenerative medicine. Interestingly, karyotypic aberrations found in stem cell lines have also been observed in tumours. The identification of mechanisms behind the development of such chromosomal aberrations could help to maintain the stability of stem cells, and therefore provide the safe use of stem cells for regenerative medicine, as well as providing scientific knowledge about mechanisms behind cancer development.
This literature review will focus on cultural adaptation in mammalian cell lines, using stem cells as explanatory example to address the processes behind this phenomenon.
Culture adaptation in stem cells
Undifferentiated human SC are used to derive embryonic stem cells (ESC), and induced pluripotent stem cells (iPSC) (Takahashi et al., 2007; Thomson et al., 1998). The following figure will provide a general overview about the derivation of human ESCs (hESCs) and human iPSCs (hiPSCs) and their use and importance in clinical approach.
Figure 1: Derivation of hESCs and hiPSCs from hPSCs and their clinical importance (modified by Dakhore et al., 2018). (a) Source: a brief overview about the derivation pathway of hiPSC and hESC from their original source hPSC. hESC, derived from the inner cell mass of preimplantation blastocysts, remain undifferentiated when isolated in culture, but at the same time keep their pluripotency under appropriate culture conditions (Martin, 1981; Martin and Evans, 1975). Owning the ability to differentiate into cells of three different germ layers (mesoderm, ectoderm, and endoderm) (Takahashi et al., 2007; Thomson et al., 1998), directed differentiation allows the transformation of hPSC into specific organ tissues with a high degree of efficiency in vitro (Hirschi et al., 2014). Reprogramming of somatic cells, by ectopic expression of the 4 genes Oct4, Sox2, Klf4 and cMyc (Cai et al., 2015), gives rise to hiPSCs through activation of transcription factors characteristic for pluripotency (Figure 1), thus generating unlimited supply. (Takahashi and Yamanaka, 2006; Takahashi et al., 2007). (b) Raw material: an illustration of the isolation of the obtained cells from the original source and possible occurring risks to underline the importance of genomic quality maintenance. (c) Final product / applications: possible clinical use of gained cells after cell culture and their next differentiation steps. The broad use of SC underlines the importance of genomic maintenance and indicated disastrous outcomes though culture adaptation of cell lines in therapeutic approach.
Mutations found in SC derived culture adapted cell lines influence the process of self-renewal and proliferation. Self-renewal is the mechanism by which stem cells divide symmetrically or asymmetrically to create more cells ensuring perpetuation to regenerate adult tissues. Proliferation and self-renewal are two distinct processes, although both mechanisms affect cell division. The more general term proliferation refers to the cell division of all types of stem and progenitor cells among others, while self-renewal describes the development of at least one daughter cell identical to the mother cell, thus maintaining their pluripotency and undifferentiated state.
However, defects in the self-renewal mechanism can lead to developmental defects, such as chromosomal aberrations (He et al., 2009). Chromosomal aberrations result in abnormal gene expression and lead to karyotypic instability, which creates instable phenotypes (Drubin and Hyman, 2017).
Figure 2: Pathways of Stem Cells and the influence of environmental factors (modified from Avery et al., 2013). Stem cells, in order to multiply themselves, can undergo either the process of differentiation, self-renewal or apoptosis. Selective pressures are assumed to supress the pathway of differentiation, favouring self-renewal (Avery et al., 2013).
For clinical use, the identification of occurring changes is of priority, thus leads to the necessity of continuous monitoring, which is costly, laborious and extreme time consuming.
Identification of Culture Adapted Cells
Until the complete and certain elimination of occurring karyotypic abnormalities in mammalian cell lines, continuous monitoring of the genomic integrity is important to maintain the culture quality (Howe et al., 2014), as malignant alterations might affect the developmental potential of those cells (Lund et al., 2012). So far, conventional karyotyping (G-banding) is the most frequent used method to observe the genomic condition of the cells (Lund et al., 2012; The International Stell Cell Initiative, 2012). However, this method is slow, cost and labour-intensive, as well as limited resolution and in the number of cells to subject of analysis, where in this case it would be necessary to analyse the whole cell population (Lund et al., 2012; Yin et al., 2014). DNA array based methods are an attractive alternative, as big deletions can be also detected. However, these also appear to be costly and slow with requiring specialised expertise for the data analysis (Everett et al., 2010; Lund et al., 2012).
Compared to G-banding techniques a small improvement regarding detection rate was seen using Fluorescence in situ hybridisation (FISH). On the other hand, detection of aneuploidy can be strongly influenced by cellular phenomena and hybridization artefacts leading to difficulties in the interpretation of results. Both methods, G-Banding and FISH and be combined to get a better detection rate, as Saaren et al. (2009) showed (Table 1). This revealed the emergence of trisomy 7 and trisomy 19 in human neural progenitor stem cells.
However, the use of standard cytogenetic and interphase FISH techniques was suggested to require a level of aberrated cell in cell culture between 0.2 and 5 % (Olariu et al., 2010).
Random amplification of polymorphic DNA (RAPD), a multiplex PCR-based molecular system, is another efficient DNA alteration, identification method (Singh et al., 2006), but data for long-term culture-induced induced variations were seen to be insufficient. This was refuted by Pavlova et al. (2015) by using primer P447, which demonstrated culture stability in human adipose derived stromal cells for not more than 5 passages.
To overcome most of those disadvantages, another strategic approach named KaryoLiteTM BoBsTM, a screening tool to facilitate molecular karyotyping, was shown to be faster and easier in comparison to previously mentioned techniques (Kong et al., 2017; Pérez-Durán et al., 2015; Vialard et al., 2011). It is a bead-based suspension, low density array technology, with a very high sensitivity for detecting aneusomies.
[Fehlen hier noch typische detections methoden? In drosophila is das drin! Das kannst du damit vergleichen was schon gemacht wurde]
Figure 2: The assay design of KaryoLite BoBs (PerkinElmer, 2011). The top part of the figure shows a metacentric chromosome as an example for demonstrating the concept behind KaryoLite Bobs. Each of the beads (pink) has three different BAC clones on each bead type, which refer to three neighbouring regions on the chromosome. This increased binding site results in averaging of the signal and thus, minimising the effect of allelic losses during the PCR reaction (allelic dropouts). Two beads are used per chromosome arm (except of acrocentric chromosomes), which provides information about the dosage on telomeric and pericentromeric regions (Pérez-Durán et al., 2015; PerkinElmer, 2011).
Consistent approaches of observing culture adapted cell lines led to the identification of a number of mutations, which are likely to be provide survival advantages for these aberrated cells, thus indicating the use of those mutations as markers for faster identification for culture adapted cells.
Observed Karyotypic Changes – Identification of QTL
Chromosomal abnormalities have been reported in several different cell lines, mammalian and non-mammalian (fungi, insects, reptiles, amphibians and plants (Siegel and Amon, 2012)). Mammalian cells, which are usually diploid (except of a few species of vizcacha rats being reported to be tetraploid (Gallardo et al., 1999)), often show full gain of chromosomes (trisomy), amplifications of specific regions of alleles (copy number variations (CNVs)) or point mutations (Weissbein et al., 2017)(Figure 3). Table 1 provides an overview about the so far observed karyotypic changes in mammalian cells being mostly identified through the abovementioned detection techniques. Remarkably, many parallels between those occurrences have been reported, indicating their non-random occurrence.
Figure 3: Representative cytogenetic data from a HESC line maintained in Sheffield (Baker et al., 2007). The figure shows a g-banded karyotype of the cell line HUES 5 (passage 19) as an example for chromosomal abnormalities. The gains of chromosome 12 and 17, which are frequently seen, are here shown in form of trisomies.
Table 1: Overview of numerical and structural karyotypic changes in different cell lines with recurrent numerical and structural aberrations (modified after Ben-David and Benvenisty, 2012 and Rebuzzini et al., 2015)
Cell type
Main recurrent aberrations
Cell lines with aberrations in % References
hESCs numerical
structural gain of chr. 1, 12, 17, 20, or X
duplication 1q11q32
partial gain of chr. 12
partial gain of chr. 17
duplication 20q11.21 32-34
10-25
13
20
10 Baker et al., 2007; Brimble et al., 2004; Draper et al., 2004; Imreh et al., 2006; Inzunza et al., 2004; Lefort et al., 2008; Mitalipova et al., 2005; Spits et al., 2008; The International Stell Cell Initiative, 2012
mESCsa numerical
structural trisomy 8
trisomy 11
deletion 10qB
deletion 14qC-14qE 16
8
14
5 Gaztelumendi and Nogués, 2014; Liu et al., 1997; Luft et al., 2014; Rebuzzini et al., 2008a, 2008b
hiPSCs numerical
structural trisomy 8
trisomy 12
amplification 1q31.1
amplification or deletion 17q21.1
amplification 20q11.21
deletion 8q24.3 5
12
>25
>25
10
12 Aasen et al., 2008; Boulting et al., 2011; Martins-Taylor and Xu, 2012; Mayshar et al., 2010; Peterson and Loring, 2014
Human bone marrow-derived MSCsb numerical monosomy 13 or X
trisomy 5, 8 or 20 ≤1
15–20
Ben-David et al., 2011; Grigorian et al., 2010; Nikitina et al., 2011; Tarte et al., 2010
Human adipose-derived MSCs numerical trisomy 8 or 17
tetrasomy 11 or 17 8–12 (passage 2);
18–28 (passage 20)
Estrada et al., 2013
Human dental pulp-derived MSCs numerical
structural loss of chr. 2, 3, 5, 6, 7 8, 9 and 16
44,XX,t(13,14),-22 70
3 Duailibi et al., 2012
Suchánek et al., 2007
Murine bone marrow-derived MSCs numerical
structural clonal gain of chr. 2; non-clonal gains or losses of several chromosomes
double-minutes; chromosomal imbalances 40-70 Fan et al., 2011; Josse et al., 2010; Miura et al., 2006
Hematopoietic SCs numerical tetraploidy 13; monosomy X
46,XX,t(2;8); 45,XX,–8; 45,XX,–7,6q+ 10-15 Ge et al., 2011
EPCsc numerical 92,XXYY
+77,XX
+55,XX
gain of chr. 11 and 14 33
68
100
74 Corselli et al., 2008
Human NSCsd numerical trisomy 7 or 19
trisomy 10, 18
monosomy 18 5-24
5
5 Sareen et al., 2009
Ben-David et al., 2011
Mouse NSCs numerical clonal monosomy 17
trisomy 1
6
29 (passage 10);
62 (passage 40) Diaferia et al., 2011
Rhesus macaque PSCsf numerical
structural trisomy 17
duplication 16q
?
?
~5.4 for both Ben-David and Benvenisty, 2012
HAMSTER ?
a mouse embryonic stem cells
b mesenchymal stromal cells
c endothelial progenitor cells
d neural progenitor stem cells
f added to original table from Rebuzzini et al. 2015
The table demonstrates most of the abnormalities to reoccur in the chromosomes 1, 12, 17, 20, and X, which make up 50 % of the aberrations found in hESC lines (Rebuzzini et al., 2015; The International Stell Cell Initiative, 2012). The most highlighted findings in those cells are duplications of 1q11q32, whole or partial gain of the chromosomes 12 and 17, as well as the duplication of the 20q11.21 region and aneuploidy of chromosome X (Table 1) (Baker et al., 2007; Brimble et al., 2004; Draper et al., 2004; Imreh et al., 2006; Inzunza et al., 2004; Lefort et al., 2008; Mitalipova et al., 2005; Spits et al., 2008; The International Stell Cell Initiative, 2012). Especially the gain of specific short nucleotide polymorphisms (SNPs) indicate the copied genes to provide the selective and proliferative advantages, as well as resistance to apoptosis observed in those cells rapidly taking over the culture (Baker et al., 2007; Lefort et al., 2008; Spits et al., 2008; The International Stell Cell Initiative, 2012). The following chapter will highlight some of those mutations to give insights about the involved genes and also demonstrate suspected parallels to cancer cells.
Chromosome 17 in stem cells
Chromosome 17 is the largest of the human chromosomes and associated with a wide range of human genetic diseases, as it contains genes like BRACA1, encoding for early-onset breast cancer localised on 17q (Ford et al., 1998), and TP53 encoding the p53 protein responsible for DNA damage response (Zody et al., 2008) localised on 17p13.1 (Shlien et al., 2010). [Wo ist die verbindung zu den vermerten regionen??]
Overlapping with the data from Baker et al. (2007), which reported the gain of region 17q25-qter in hESC (Figure 4), indicating an amplicon in the terminal region of chromosome 17, Kraggerud et al. (2002) and Łastowska et al. (2002) state a similar regions, 17q24-qter and 17q23.1–qter to be amplified in testicular germ cell tumours (TGCTs) neuroblastomas, which the most common extra-cranial solid tumour in infants and children (Colon and Chung, 2011).
The gene BIRC5, located on 17q25.3 (NCBI, 2019), encoding for Survivin, a protein that inhibits apoptosis and regulates cell division (Chiou et al., 2003), was found to be highly expressed in hES and teratomas (Blum et al., 2009), and therefore is likely to represent a candidate gene, providing resistance to apoptosis. Interestingly, Blum et al. (2009) found BIRC5 to be downregulated in embryonic bodies, which are aggregates formed in suspension by pluripotent stem cells (PSC). The formation of embryoid bodies is commonly used to produce different cell lines and can thus be the origin of cell lines (Rungarunlert et al., 2009). Blum et al. (2009) therefore suggested other oncofetal genes normally expressed in hESCs, to promote tumour formation. [das noch mal verstehen]
mESc are more often seen to develop alterations in the number of chromosomes, with reoccurring chromosome 8 and 11 trisomy. The distal portion of the the q arm of chromosome 11 in mESCs shows high similarity to the human chromosome 17 (Liu et al., 1997; Rebuzzini et al., 2015), thus indicating the occurrence of aberrations being non-random.
Figure 4: Illustration of CNV of region 17q25-qter (modified from Avery et al., 2013; Larkin et al., 2000). The illustration shows 2 chromosome 17 pairs, the wild-type on the left and the mutated variant on the right. The yellow bubble indicates the centromere. The red marks on the wild-type chromosome show the position of region 17q21-qter. The left q-arm of the variant shows the copy of 17q21-qter.
HAVE I MENTIONED IN WHICH CELLS?
Homogeneous staining region
In 1976 G-banding, short for Giesma-banding, analysis of metaphase chromosomes in differentiated cells (WHAT KIND OF CELLS? Stem?) revealed heretofore homogeneous staining regions (HSRS). First named by Biedler and Springer (1976), HSRS, today also abbreviated HSR, are terminal or interstitial additions to a chromosome with various lenths commonly staining uniformly with banding techniques. They are cytogenetic indicators of gene amplification and are of prognostic importance as they are frequently found in a variety of solid tumors (Lee et al., 2009; Samaniego et al., 1990).
Baker et al. (2007) to my knowledge first documented HSR occurrence in HESCs, with the amplification originating from region 17p11.2 in the adapted HESC subline H14 investigated through the use of CGH and FISH. Upregulated candidate genes in HSR-containing H14 cell line, analysed by microarray study comparing those to its normal karyotype, were found to be TOP3A, COPS3 and MAPK7 (Baker et al., 2007), which are documented as potentially oncogenic in osteoscaromas (Van Dartel and Hulsebos, 2004).
Derived from the same region an abnormal triplication of this segment was found in the HESC subline Shef5. Furthermore, five cases of HSR have been documented in TGCTs in which one of those the abnormality also derived from region 17p11, whereas in the second case the abnormality originated from 1p32 (Samaniego et al., 1990). The same study also showed HSR only occurred after 15 subcultures, which points to development in late passage numbers.
Chromosome 12 in stem cells
The karyotypic changes in chromosome 12 of stem cells is mostly observed as gain of the whole chromosome (trisomy), which is more frequently reported than trisomy of chromosome, and also in form of an isochromosome 12p (Baker et al., 2007). i(12p), detected as sole abnormality on Baker et al. (2007), is also a characteristic karyotypic marker for TGCTs (Bosl et al., 1994). In TGCTs the gain of material from chromosome 12p is seen in from of 2 amplicons, 12p11.2-12 (Mostert et al., 1998; Rodriguez et al., 2003) and 12p13 (Korkola et al., 2006). Mayshar et al. (2010) identified amplicon 12p13 also in a culture adapted induced pluripotent stem cell (iPSC) line.
Potential candidate genes for 12p13 are CCDN2, CD9, GAPD, GDF3, NANOG, and TEAD4, as they were shown to be highly over-expressed (Skotheim et al., 2006). Induced overexpression of NANOG, known as transcription factor essential to maintain the phenotype of pluripotent ESC, prevents hESCs from differentiation (Darr, 2006). However, the increase of NANOG was not detected in all observed culture adapted cell lines, which points to a variation of possibilities for ES cells to adapt to culture.
[hier noch andere paper als (Harrison et al., 2012)]
Due to the results listed in table 1, the predicate of Draper et al. (2004), selective advantages arising through the increased dosage of genes located on chromosomes 17q and 12 is very likely. However, more genes are seen to be involved in the process of cultural adaptation and it still seems to be unclear which the exact causative mutations are. Clearly, parallels can be drawn to the development of cancer, as the same aberrations are found in both cell types, SCs and cancer cells, thus leading to very similar phenotypes like proliferation and self-renewal [achtung hier noch mehr details, dass hast du noch nicht genannt] [PAPPER]
Chromosomal aberrations in other cell lines than stem cells
rmPSC
To my best knowledge, the analysis of rhesus macaque PSCs (rmPSCs) regarding karyotypic changes occurring through cultural cell adaptation was so far only once carried out by Ben-David and Benvenisty (2012). The low number of occurrence of aberrations in of approximately 5.4 % in rmPSC (Table 1) might be attributed to the low number of passages of the examined cell lines. Chromosme 17, which showed a full trisomy, is synthetic to the human chr. 13, as well as chr. 16, with the mutation expressed as duplication of its q-arm, being synthetic to the human chr, 17 (Rhesus Macaque Genome Sequencing and Analysis Consortium, 2007).
[hier noch weitere einfuegen]
WHAT ABOUT SNPs??? = genetic markers
What about linkage disequilibrium?
Quantitative trait loci (QTL), regions of DNA or marker intervals being associated with specific phenotypic traits, for culture adaptation, seem to be better explored in organisms like Drosophila and Arabidopsis. However, obtaining causative genes or nucleotides within genes (QTN) appears to be very problematic in mammalian, as well as non-mammlian cells, due to various reasons – environmental factors?? Which will be illuminated under paragraph X (Erickson et al., 2004)
STATE THE DIFFERENCES OF FINDINGS!!!
Frequency and passage related occurrence through culture adaptation
Passage numbers
The higher the number of passages, the more frequent abnormalities have been reported (Baker et al., 2007; The International Stell Cell Initiative, 2012) (Figure 5). The passage number of a cell culture is the record of the number of times the cells from one culture have been sub cultured a statistical technique used to understand the impact of risk in complex systems flasks’ (European Collection of Authenticated Cell Cultures and Public Health England, 2017). Between passages, cells have the potential to undergo three divisions, self-renewal, mutation, or death/differentiation (Olariu et al., 2010).
Figure 4: Illustration of culture adapted cells taking over the culture with rising passage number (modified from Avery et al., 2013).
To evaluate the impact of passaging cell lines, Olariu et al. (2010) executed a Monte Carlo simulation (MCS), which is a statistical technique used to understand the impact of risk in complex systems (Tenekedjiev et al., 2011). Their results show the first detected aberrations to appear at the 0.2% level of abnormal cells in culture after 14 passages on average, with a complete takeover of the culture after 42 passages (Figure 5), thus which confirming the results from other studies comparing the number of mutations occurring in early and late passages (Table 1). However, an accurate and generally valid prognosis cannot be derived from the results of the MCS due to significant deviations. For example, some 0,2 % variants already appeared after 8 passages, while others only appeared after 21 passages. Additional 25 were required until the total culture was populated with abnormal cells, although deviations were seen in these numbers as well.
Figure 5: Passage numbers and exponential growth of culture adapted cells (Olariu et al., 2010). Olariu et al. (2010) assumed values for S = 2.0, pm = 10-6 and N = 106 (S = selective advantage / the ratio between the presumption of variant and wild-type cells surviving as stem cells postdivision: N = number of genetically homogenous embryonic stem cells (ESC); pm = frequency of occurring variant ESC). Simulation were run 50 times, whereby the passage number, at which the proportion of abnormal cells reached 0.2, 5, 50 and 90% of all cells in the population, was recorded in each run. The illustration showed the average time for mutant cell appearance at those levels across the 50 runs. The earliest and latest passages at which variant cells reaches each detection level are indicated.
The exact set in of karyotypic change (the number of passage) cannot be ascertained, as this seems to vary in different studies. This indicates causative mechanisms behind cultural adaptation to differ between the laboratories, where the mutations were observed (see paragraph ‘Possible mechanisms behind culture adaptation’). However, long-term passaging was found to put cells under high levels of stress, which could therefore be a reason for the development of genetic changes.
Selective advantage
Olariu et al. (2010) carried out further tests, mixing established culture adapted cells with their diploid paternal equivalent, to determine the selective advantage accounting for the observed cases of cultural adaptation. The achieved data were compared with their numerical simulations. Pm was set as zero with N matching the number of passages. The variation of S in the simulation indicated a selective advantage between 2.0 and 2.8 being a close convergence to the rate of variant takeover in the mixed cultures tested. Compliant to the suggestion of cultural adaptation being a dynamic process, the results showed selective advantages to be higher in later passages. An overview about the results, including details about the methods used during the analysis is to find in Figure X. Although, due to the consistent factors within the process of adaptation between hES and EC cells I tumours, intrinsic factors of pluripotent cells are likely to play a role, the frequent occurrence of S = 0.2 indicated a minimum value that necessary to be reached in order to be recognised as a mutation and thus to develop the selective advantage (Olariu et al., 2010).
Figure 6: The rate of takeover of culture adapted cells over normal, diploid cells (Olariu et al., 2010). (A) The figure illustrates the results from experiments mixing 99% diploid hES with 1% of karyotypically abnormal cells derived from the same line as the diploid hES they are mixed with. The different lines are indicating the single experiments with reference to the passage it was taken from, as well as the used culture strategy (T = trypsine/versine; C = collagenase and scraping). The number of abnormal cells was controlled by G-banding cytogenesis techniques and at each passage 106 cells were seeded. Simulation data for each experiment was adjusted for the calculated value S, which is also indicated in the plot. (B). Obtained data from the University of Sheffield and Buzzard et al. (2004), the graphs show cases in which the sequential stages of culture takeover by abnormal cells have been recorded. The passage numbers on the X aches are taken post initially to the detection of the variant. The mid-passage H7.s6 takeover from figure X is also shown in this graph for better comparison (N = 106, S = 2, and pm = 0).
Possible mechanisms behind culture adaptation
For summary/to put the information given into context, certain changes in the genome of cell lines appear when cells are transferred from a living tissue into culture. The changes are mostly reoccurring gains of specific genes, encoding for those factors needed to survive the selective pressures a transferred cell is confronted with in its new environment. Thus, in this evolutionary process, certain cells develop selective advantages, while cell maintaining their genetic code are subject to apoptosis. The reason for certain cells to develop these changes, while others don’t, seems to be unclear. However, for researchers is it is important to develop strategies to bypass this mechanism, and therefore to understand the causative factors.
Mutation and selection (?)
The mechanism(s) behind cultural adaptation of cells must involve two events: mutation and selection (Olariu et al., 2010). It is important to understand both, to not only eliminate aberrations but also to gain scientific knowledge for the development of treatments (Olariu et al., 2010).
In non-mammalian cells like Drosophila (Orozco-Terwengel et al., 2012), this process seems to be well understood, whereas for mammalian cells some aspects still seem to be unclear.
Population size
Olariu et al. (2010) exchanged the values N, pm or S from the Monte Carlo Simulation, used to plot the passage numbers in relation to the percentage of abnormal cells, in order to examine the dependencies of the values more closely. The results lead to the conclusion that a reduced population size in vitro may lead to more stable cultures with lower risk of emerging aberrations, than those including a larger transfer of cells, as seen in mass culture techniques using enzymatic harvesting (Olariu et al., 2010).
Figure X: Population size in relation to occurrence of mutations and duration of appearance (Olariu et al., 2010). As fewer cells being subject to a mutation, a decrease in pm was shown to lead to a decrease in N. At the same time, the duration of occurrence of abnormal cells in culture rose exponentially as the population size was reduced. For N between 106 and 5×104, the probability of cultures reaching a level of 5 % of mutated cells was shown to be high (>0.9) by passage 50, whereas a reduction of N to 103 extended the passaging time up to 400 passages for abnormal cells until they reached the 5 % level with a probability higher than 0.9 (Olariu et al., 2010). (A) shows the probability of mutant population reaching 5 % when N = 103-106. (B) illustrates the passage number at which the probability of 5% mutant cell appearance is 0.5 when N=103–106. (C) The probability of the mutant population reaching 5% when pm=10-3–10-10. (D) The probability of the mutant population reaching 5% when S=1–3. Simulations were run 50 times for each set of parameters.
The results were further confirmed by a direct comparison using one large culture of 104 of initial cells, and 10 cultures each containing initially 103 cells. After 500 simulation runs the rate of mutation appearance was higher in single culture compared to the smaller ones.
Figure X: The impact of population size on cultural cell adaptation (Olariu et al., 2010). ‘With N = 104, pm = 106, and S=2, a level of 5% abnormal cells was reached with a probability of 0.5 after 58 passages when maintained as a single culture, but only after 66 passages when maintained as 10 smaller cultures’ (Olariu et al., 2010). With reaching the 50 % Level of adapted cells the imbalance became more marked as 70 passages were reached with a probability of 0.5 for the big, single culture, but 235 passages for the 10 small cultures. The results are illustrated in Figure X. Increasing pm led to the increase of the probability of abnormal cultures and a decrease in S had the impact of decreasing the probability of abnormal cultures. If 1.0 (i.e. no selective advantage) was set for S, the probability of abnormal cell cultures approached 0. (A) Simulation for 104 cells being maintained over sequential passages as single population or in 10 smaller populations 103, showing the probabilities of cultures reaching 5%. (B) Simulation for 104 cells being maintained over sequential passages as single population or in 10 smaller populations 103, showing the probabilities of cultures reaching 50%. The illustration summarises the results of 500 simulation runs in both cases.
OTHER PAPERS ON POPULATION SIZE!!! + vergleich/ evtl kannst du das alles kuerzen – was ist das genaue ergebniss? Was sagen die anderen? Das vergleichen und weniger datrn ?
However, as the calculations are pure simulations considering only a few variables, the data might not be valid for
Considering ony a few variables , this is only an simulation  is it really only calculations? They did actually test that ?
Oxygen levels in culture
Noch mal paragraph REACTIVE Oxigen species angucken von (He et al., 2009)
More likely, the stressors causing aberrations in hESCs are due to altered living conditions in culture. The physiological environment of stem cells in vivo comprises approximately 1-5% O2, depending on the tissue. Much higher oxygen levels are found in the atmosphere of stem cells in media, containing about 20% O2 (Li and Marbán, 2010). In 2007 Baker et al. (2007) suggested high oxygen tension might promote shortening of the telomeres and therefore cause chromosomal damage [Paper wenn mehr info gewollt: (Von Zglinicki et al., 2000)]. Low oxygen tension (hypoxia) was shown in 2018 to contribute critically to pluripotency of human embryonic stem cells (Dakhore et al., 2018). Furthermore, it was also shown that reactive oxygen species (ROS) influence the genomic stability in cardiac and embryonic stem cells, as karyotypic abnormalities in primary human cardiac stem cells were repressed in culture containing 5% of oxygen. An increase in aneuploidy was developed by addition of antioxidants (Li and Marbán, 2010). Li and Marbán (2010) showed that the addition of low concentrated antioxidants has the ability to decrease DNA damage in cardiac stem cells, but potentiated damage when supplied in high concentrations in decreasing the levels of DNA repair enzymes. However, the exact impact of oxygen levels regarding the occurrence of chromosomal aberrations still remains unclear.
oxygen
They use glycolosys metabolism normally now they use oxidative phosphorylation –
Poor Mitotic Control
The rapid proliferation of hPSCs, their inefficient cell-cycle checkpoints (Desmarais et al., 2012; Filion et al., 2009; Weissbein et al., 2014),
Decantination checkpoint G2
As described above, most of the karyotypic changes involve trisomy. According to Baker (2007) this points to hESC being error prone for chromatid separation during mitosis, as the disruption of the G2 decantenation checkpoint can lead to aneuploidity (Wenzel and Singh, 2018). This checkpoint delays entry into mitosis from G2, if the chromosomes have not been sufficiently disentangeled or decantenated (Damelin et al., 2005). Damelin et al. (2005) found this process in mESCs to be highly inefficient. However, the checkpoint efficiency improved in ESs as they were induced to differentiate, thus indicating the deficiency might be a feature of the undifferentiated state (Damelin et al., 2005). According to the results of Damelin et al. (2005), a deficiency in the decantenation checkpoint is expected to increase the rates of chromosomal aberrations in both stem cells and cancer stem cells.
Mitotic spindle checkpoint
The mitotic spindle checkpoint is a surveillance mechanism to delay anaphase onset until all chromosomes are aligned correctly in a bipolar manner to the mitotic spindle (May and Hardwick, 2006). Defects in this mechanism contribute to chromosomal instability and aneuploidy. The occurrence of disordered chromosomes during mitosis inhibits the ubiquitin-ligase activity of the anaphase-promoting cyclosome, which leads to the prevention of early chromosomal segregation and thus supports the distribution of the genetic material (Bharadwaj and Yu, 2004).
Nonetheless, next to culture induced genetic changes, epigenetic aberrations can also influence the genomic stability of cells.
DNA methylation aberrations??? Weissbein 2017
Condensation effects?? Lamm 2016
Lamm et al. (2016) show aneuploid hPSCs to have altered levels of actin cytoskeletal genes which are controlled by the transcription factor SRF. SRF rescues impaired chromosome condensation and segregartion defects in affected cells (Lamm et al., 2016).
Furthermore, SRF downregulation in diploid hPSCs induces replication stress and perturbed condensation similar to that seen in aneuploid cells. Together, these results suggest that decreased SRF expression induces replicative stress and chro- mosomal condensation defects that underlie the ongoing chromosomal instability seen in aneuploid hPSCs. A similar mechanism may also operate dur- ing initiation of instability in diploid cells.
Overcoming Cultural Cell Adaptation
Maintaining the homologous state was to my best knowledge not yet achieved in mammalian cells. However, techniques for correction of culture adapted cell lines have already been developed.
BH3 Mimetics
In 2018, Cho et al. observed late passage numbers of the hESC subpopulation YM155, a survivin inhibitor, and found them to be highly resistant to diverse cell death stimuli. YM155-resistant (YM155R) cells showed a high expression of BCL2L1, as well as a matching gene signature with the Cancer Therapeutics Response Dataset, indicating them to be selectively ablated by BH3 mimetics (Cho et al., 2018). BH3 mimetics, small molecules mimicking BH3-only proteins, are a class of drugs directly activating apoptosis by binding and inhibiting the prosurvival members of the BCL-2 family of proteins (Baell and Huang, 2002; Delbridge and Strasser, 2015). Overexpressed BCL-2 functions to inhibit apoptosis (Baell and Huang, 2002). Treatment of YM155R with sub-optimal dose of BH3 mimetics led to spontaneous death. Additionally, they found YM155-sensitive to maintain its pluripotency after the treatment. Therefore, Cho et al. (2018) declared the use of BH3 mimetics as a promising strategy to eliminate hESC carrying a selective survival advantage.
[delbridge and Strasser pic einfugen!!!!!! Zu bh3 mimetics workprocess vllt kannst du dann oben bishen kuerzen und unten extenden]
BH3 mimetics in anderen mammals?
Genome Editing
Adikusuma et al. (2017) achieved the effective deletion of a whole Y chromosome using CRIPSR/Cas9, demonstrating the application of gene editing for targeted correction of both in vivo and in vitro. They envisage the strategy to be used in modelling of aneuploidy syndromes, thus indicating its potential to correct chromosomal aberrations in stem cells (Adikusuma et al., 2017). To my best knowledge, the specific correction of culture adapted stem cell lines was not yet attempted. However, gene editing techniques were already performed successfully in stem cells for correction of point mutations and SNPs (Bak et al., 2018; Flynn et al., 2015; Turan et al., 2016), next to full chromosome deletions as seen above (Adikusuma et al., 2017). However, gene editing is still costly and therefore, the correction of culture adaptation induced karyotypic changes, more specifically the rescue of adapted cell lines has to be considered carefully.
Potential clinical use for mammalian culture adapted stem cell lines
REASONS FOR STEM CELLS AS MODEL ORGANISMs
Drubin and Hyman (2017) argue, stem cells, including induced pluripotent stem cells (iPSC), embryonic stem cells (ESC), and somatic stem cells (SSC), can be used as such model organism, as they are known to combine many advantages of tissue culture models.
Early passage stem cells derived from healthy tissues have usually a normal physiological state and have the advantage to lack abnormalities that are typical for other cell culture lines like HeLa cells. The potential of stem cells to differentiate into many different cell types with reliable detection of their phenotypic differences, which can be studied in isogenic cells derived from a parental cell line (Noggle et al., 2005), make stem cells a considerable candidate for studying problems in molecular and cell biology and cancerogenesis (Drubin and Hyman, 2017). Especially the investigation of fundamental mechanism can be rapidly translated into understanding diseases due to their differentiation ability. Additionally, the powerful tool CRISPR/Cas9 can be successfully used to edit stem cells, allowing the observation of impact of different mutations in different cell cultures and tissues (Drubin and Hyman, 2017).
Comparing genome editing and stem cells as a model system
HeLa cells
Biomanufacturing
Rapid cultural adaptation can facilitate the evolution of large-scale cooperation
https://www.cell.com/molecular-therapy-family/methods/fulltext/S2329-0501(18)30045-7
Aneuploidy-specific therapies
Numerical and structural chromosome abnormalities are a hallmark of cancer (Gordon et al., 2012). Rearrangements are known to have a big impact on tumour development by inactivating tumour suppressors while activating oncogenes at the same time (Mitelman et al., 2007). On the other hand, the influence of whole-chromosome aneuploidy during tumor development is far less understood.
(Gordon et al., 2012)
Conculsion
Variant cells have been reported to often take over cultures within a few passages (Baker et al., 2007; Draper et al., 2004), thus suggesting a strong relative advantage for the adapted cells.