**2.1 Phenotype shifts and culture adaptation in hESCs**

In prolonged culture, pristine hESCs adapt to a two-dimensional, biochemically and structurally less complex, in comparison to a blastocyst, microenvironment [20]. However, the introduction into the *in vitro* culture inevitably induces selection: hESCs clones, which fit the artificial culture conditions the best, are being preferentially selected. Particularly those are clones which divide regularly with optimal doubling time, show reduced sensitivity to programmed cell death, are resistant to passaging method and cell stress arising in culture. Despite stringent culture protocols, such clones can acquire non-random chromosomal aberrations, alterations of epigenetic landscape and changes in gene expression [21–24]. Often, genes which participate in tumor formation or dissemination are involved [25], similarly to those in undifferentiated stem cell lines derived from teratocarcinomas [26].

Darwinian selection in long-term hESC cultures, accordingly, generates cells which remarkably differ from pristine hESCs. Such culture-adapted cells can obtain hazardous phenotype [27], which is similar to a malignant cancer cell line, while keeping normal levels of transcription factors (e.g. Oct 3/4 and Nanog) and other stemness-related molecular markers (e.g. SSEA-4 and Alkaline Phosphatase) [28]. Paradoxically, such significant changes in hESCs, which have acquired the adapted phenotype, can stay unnoticed if cell morphology or expression of stemness factors are not affected. Thus culture-adapted cells can escape routine quality control, which is usually based on visual evaluation or monitoring of a several selected molecular markers. Incompatible cellular alterations are quickly eliminated *in vivo*, typically by the complete rejection of the embryo. *In vitro*, however, aberrant clones may become dominant in culture and disable the safe use of the particular hESCs for clinical or biotechnological applications.

At the moment, there is no routinely applicable method which can reveal hidden shifts in hESCs phenotype or to confirm general stability of a cell culture. The golden standard for authentication of *in vitro* cell lines, e.g. ones derived from individual patients' tumors, is based on analysis of short tandem repeats (STRs). Repetitive sequences dispersed throughout the genome can provide a unique genetic profile and effectively reveal potentially misidentified cell lines [29]. However, STRs analysis is uninformative if used for specific cell culture modifications, such as co-culture of two cell types, analysis of differentiation stages within a single cell line, use of more cell subtypes derived from an individual. Similarly, techniques used for analysis of batch to batch variability, purity of cells, genome or proteome changes, as well as methods focused on a limited panel of biomarkers are not

**125**

*Intact Cell Mass Spectrometry for Embryonic Stem Cell Biotyping*

suitable for quality control in routine applications, because they are not informative

Successful implementation of pluripotent stem cells into clinical trials requires stringent assurance of the cell product quality and safety, including development of formal methodology for every step of the cells derivation and culture processes. Presence of an unwanted phenotype or deviation from the optimal state of cells in culture may inevitably result in failure of the product and a harm to a patient. Routinely used methods easily reveal microbial and viral contamination, cell misidentifications and chromosomal aberrations which occur in the culture. However, subtle phenotypic shifts or unapparent alterations cannot be detected by microscopy, narrow focused screening for molecular markers or other conventional methods. Recently, we have been working on the introduction of techniques used in bioanalytical chemistry, chemometrics and in complex data analysis into stem cell field. We have demonstrated that method of choice can be the Intact (Whole) Cell MALDI TOF Mass Spectrometry coupled with sophisticated statistics. Intact Cell MALDI TOF MS is sufficiently robust, sensitive, and cost-effective approach for monitoring of long-term cultures stability and differentiation trajectories of ESCs, therefore it has a potential to

complement the portfolio of quality control tools in clinical or bio-industrial

MALDI TOF MS employs the laser energy to desorb and ionize molecules of an analyte from the crystallized mixture with the matrix, and subsequently separates the resulting ions according to mass-to-charge (*m/z*) ratio. The organic matrix enhances energy transfer to analyte, preserves the structure of the ionized molecules, e.g. peptides, proteins or other biomolecules, and allows their precise structural analysis and identification. In cell biology, MALDI TOF MS is one of the preferred methods for proteomic analysis in a broad range of samples, such as purified or fractioned extracts of cells or tissues. The MS-based proteomics uses protein fragmentation for identification and further generation of a list of unique peptide or protein signatures in wide range of *m/z* values [34]. However, the methodological complexity and the character of the data output may limit the use of traditional proteomics in routine quality control of stem cell cultures, even if coupled with transcriptomics or (meta)genomics. Even when the intact (whole) cells are used as an analyte, MALDI TOF MS can generate rich spectra without the need of previous cell lysis, fractionation or protein extraction. Mass spectra contain signals for small proteins and peptides, and a variety of other low-mass molecules, including metabolites. Analysis of specific spectral (peak) signatures has been successfully introduced to clinical microbiology, where MALDI TOF MS enables the rapid discrimination, or "biotyping", of bacterial species without the necessity of complex sample processing [35, 36]. Generally the same approach - utilization of relevant spectral patterns as inputs for further processing and analysis [33] - can be used for discrimination of cancer cells [37, 38] or abnormal stem cells in long-term cultures, even in high-throughput setup [39, 40]. Intact Cell MALDI TOF MS was used to identify spectral signatures of glial cells and their classification to astrocyte, microglia and oligodendrocyte type [41]. Principal component analysis then revealed informative peaks for deeper spatial analysis using mass spectrometry imaging in whole brain sections. Similarly, mass spectra have demonstrated to contain sufficient information to reveal the immunophenotype and activation state of immune cells, [42–45] or to classify distinct mammalian cell lines [46, 47]. Moreover, MS can reveal changes associated with molecular phenotype, which occur within cell lines and sublines of common genetic

*DOI: http://dx.doi.org/10.5772/intechopen.95074*

and often are laborious and costly [30–32].

applications [33].

**3. Intact cell MALDI TOF MS**


#### **Table 1.** *Risk assessment in clinical grade hESCs culture.*

#### *Intact Cell Mass Spectrometry for Embryonic Stem Cell Biotyping DOI: http://dx.doi.org/10.5772/intechopen.95074*

*Mass Spectrometry in Life Sciences and Clinical Laboratory*

**2.1 Phenotype shifts and culture adaptation in hESCs**

other culture parameters (**Table 1**) [18, 19].

for clinical or biotechnological applications.

• microbial and viral contamination • cell line cross-contamination

• karyotype instability

• carry-over of animal products in culture

*Risk assessment in clinical grade hESCs culture.*

• activation of oncogenes and risk of tumorigenicity in patient • acquisition of immunogenicity and risk of graft rejection in patient

• failure of differentiation into functional phenotype

teratocarcinomas [26].

(e.g. bFGF, activin) may be a solution, if combined with rigorous quality control of

In prolonged culture, pristine hESCs adapt to a two-dimensional, biochemically and structurally less complex, in comparison to a blastocyst, microenvironment [20]. However, the introduction into the *in vitro* culture inevitably induces selection: hESCs clones, which fit the artificial culture conditions the best, are being preferentially selected. Particularly those are clones which divide regularly with optimal doubling time, show reduced sensitivity to programmed cell death, are resistant to passaging method and cell stress arising in culture. Despite stringent culture protocols, such clones can acquire non-random chromosomal aberrations, alterations of epigenetic landscape and changes in gene expression [21–24]. Often, genes which participate in tumor formation or dissemination are involved [25], similarly to those in undifferentiated stem cell lines derived from

Darwinian selection in long-term hESC cultures, accordingly, generates cells which remarkably differ from pristine hESCs. Such culture-adapted cells can obtain hazardous phenotype [27], which is similar to a malignant cancer cell line, while keeping normal levels of transcription factors (e.g. Oct 3/4 and Nanog) and other stemness-related molecular markers (e.g. SSEA-4 and Alkaline Phosphatase) [28]. Paradoxically, such significant changes in hESCs, which have acquired the adapted phenotype, can stay unnoticed if cell morphology or expression of stemness factors are not affected. Thus culture-adapted cells can escape routine quality control, which is usually based on visual evaluation or monitoring of a several selected molecular markers. Incompatible cellular alterations are quickly eliminated *in vivo*, typically by the complete rejection of the embryo. *In vitro*, however, aberrant clones may become dominant in culture and disable the safe use of the particular hESCs

At the moment, there is no routinely applicable method which can reveal hidden shifts in hESCs phenotype or to confirm general stability of a cell culture. The golden standard for authentication of *in vitro* cell lines, e.g. ones derived from individual patients' tumors, is based on analysis of short tandem repeats (STRs). Repetitive sequences dispersed throughout the genome can provide a unique genetic profile and effectively reveal potentially misidentified cell lines [29]. However, STRs analysis is uninformative if used for specific cell culture modifications, such as co-culture of two cell types, analysis of differentiation stages within a single cell line, use of more cell subtypes derived from an individual. Similarly, techniques used for analysis of batch to batch variability, purity of cells, genome or proteome changes, as well as methods focused on a limited panel of biomarkers are not

**124**

**Table 1.**

suitable for quality control in routine applications, because they are not informative and often are laborious and costly [30–32].

Successful implementation of pluripotent stem cells into clinical trials requires stringent assurance of the cell product quality and safety, including development of formal methodology for every step of the cells derivation and culture processes. Presence of an unwanted phenotype or deviation from the optimal state of cells in culture may inevitably result in failure of the product and a harm to a patient. Routinely used methods easily reveal microbial and viral contamination, cell misidentifications and chromosomal aberrations which occur in the culture. However, subtle phenotypic shifts or unapparent alterations cannot be detected by microscopy, narrow focused screening for molecular markers or other conventional methods.

Recently, we have been working on the introduction of techniques used in bioanalytical chemistry, chemometrics and in complex data analysis into stem cell field. We have demonstrated that method of choice can be the Intact (Whole) Cell MALDI TOF Mass Spectrometry coupled with sophisticated statistics. Intact Cell MALDI TOF MS is sufficiently robust, sensitive, and cost-effective approach for monitoring of long-term cultures stability and differentiation trajectories of ESCs, therefore it has a potential to complement the portfolio of quality control tools in clinical or bio-industrial applications [33].
