**5. Applications of intact cell mass spectrometry in quality control of embryonic stem cell cultures**

Monitoring of clinical-grade stem cells during manipulation, banking or quality control by appropriate tools is the essential prerequisite for their application. We hypothesized that different cell and tissue types or their different states may vary in levels of numerous small molecules, metabolites or peptides and proteins. An unambiguous and unbiased chemical fingerprint obtained by MS can thus reflect such divergences with high sensitivity. In addition, spectral patterns can serve as a highly informative input for subsequent statistical analysis and classification.

To test this hypothesis we used a mouse model of primary hyperoxaluria I - a congenital disorder that affect enzymatic machinery of glyoxylate metabolism. Primary hyperoxaluria I causes oxalate deposits to localize in liver and kidneys, and ultimately lead to hepatorenal failure and extrarenal manifestation of the disease. Alterations of chemical composition within the tissue microenvironment of hyperoxaluric animals can be translated into specific patterns in mass spectra. A dataset, composed of peaks and their corresponding intensities obtained from diseased and healthy animals, was used as an input for cluster and classification analysis and machine learning (ANN) prediction. Spectral patterns clearly distinguished samples from healthy and hyperoxaluric animals and, in parallel, the ANN correctly predicted the category based solely on mass spectrum fingerprint [65].

Generally the same approach can be used for rapid discrimination of cells occurring in stem cell cultures. Mass spectra from pure populations of mESCs, hESCs, and mouse embryonic fibroblasts (MEFs) contain enough information to distinguish the cell types by cluster analysis. Interestingly, these spectral profiles are not lost even in case of mixed populations of two cell types, such as in crosscontaminated cell cultures. Therefore, they can serve as a basis for quantitative estimation of the individual cell types in the mixture. To model such scenario, a broad panel of binary suspension mixtures containing hESCs and MEFs or hESCs and mESCs in defined ratios was prepared. Mass spectra were recorded, processed and the spectral patterns assigned to known quantities of cells in suspension. Resulting dataset then represented calibration data matrix, suitable for quantitative prediction by ANNs. Indeed, well-trained and validated network predicted the number of contaminating cell type with high accuracy in otherwise homogenous and morphologically uniform mixtures. The precision of prediction was dependent on a number of peaks and corresponding peak intensities included in the data matrix. The apparent, dominant peaks with high intensity, did not contribute significantly to the classification. Their exclusion had a negligible effect on the ANN performance, suggesting that there is rather a cumulative effect of minor, but informative peaks, essential for the analysis output [53].

In the long term cultures of hESCs, abnormal clones with the origin in pristine hESCs are inevitably being generated, selected and, finally, expanded. The CCTL 14 hESC line, which has been propagated over hundreds of passages *in vitro* suffers

#### **Figure 4.**

*Monitoring of differentiation of hESCs into early lung progenitors (ELEPs) by intact cell MALDI TOF MS. hESCs induced to ELEP differentiation lose gradually the gene signatures associated with pluripotency and acquire the phenotype of lung progenitors. Differentiation trajectory of cells goes through several immature stages and reaches the final, differentiated stage approximately at day 21. Samples for intact cell MALDI TOF MS were collected prior to induction of differentiation (hESCs) at days 1, 3, 8 and 10 (D1, D3, D8, D10) and finally at the final stage (ELEPs). The lung cancer cell line A549 was used as lung-associated, but abnormal sample class. Cluster analysis, such as PCA or a dendrogram, based only on spectral patterns shows clear separation of classes and indicate that ELEPs represent indeed a distinct cellular entity [66].*

**131**

**Table 2.**

*grade laboratory.*

*Intact Cell Mass Spectrometry for Embryonic Stem Cell Biotyping*

from chromosomal abnormalities and shows an increased expression of CD30 and CD44 surface molecules, previously shown to be associated with acquisition of the adapted phenotype and karyotype instability [67]. In addition, adapted, high passage hESCs proliferated with reduced doubling time, and showed lower sensitivity to apoptotic stimuli and decreased capability of differentiation, than pristine hESCs. All the low (P29), mid (P72) and high passage (P269) cells maintained high and constant expression of stemness markers, e.g. Sox-2, and also share the

We recorded mass spectra from intact P29, P72 and P269 hESCs cultured under identical conditions, processed and assembled into the dataset containing 255 *m/z* values with assigned peak intensities. The mass spectra showed a high level of visual similarity, however the Pearson's correlation analysis revealed disparities in spectral patterns between P29, P72 and P269 cells. Principal component analysis then correctly discriminated P29-pristine and P269-adapted cell to individual distant cluster, while the P72-transition population cluster has been located and scattered between them. Therefore, such a robust dataset of spectral fingerprints recorded from intact hESCs contains sufficient information to distinguish normal and aberrant cells with otherwise identical morphology and expression of stemness markers. Next, we used the same approach to analyze CCTL 14 hESC line stimulated to differentiation. Generally, cell differentiation means acquisition of novel phenotypic properties, and morphological hallmarks, as well as various molecular mechanisms, involved in cell functioning. Retinoic acid (RA) is a potent inducer of embryonic patterning and neurogenesis *in vivo*, so hESCs induced to the differentiation by RA acquire the neuronal phenotype within several weeks. Mass spectra generated from cells treated with RA for only 24 hrs, contained spectral regions which allowed detection of such early differentiation events even prior to develop-

To test a routine, pre-clinical application, we used Intact Cell MALDI TOF MS to monitor the differentiation of hESCs to early lung progenitors (ELEPs). ELEPs, being the direct precursors of lung cells (pneumocytes), represent an important resource for tissue engineering, pollutant testing and pharmacological analyses. ELEPs gradually differentiate from hESCs under specific culture conditions through several stages (D1-D10). Differentiating immature cells lack an unambiguous marker, which would distinguish them from ELEPs. In addition, during differentiation process aberrant cells with unwanted properties may appear in culture. Intact

+ Discrimination of unapparent, yet critical alterations in stem or progenitor cells, which are not detectable

+ Stringent culture protocols and defined SOPs in clinical grade hESCs laboratories allow precise pre-

*Advantages (+) and limits (*−*) of Intact Cell MALDI TOF MS in quality control of hESCs culture in clinical* 

− Inherent instrumental and technical variability of MALDI TOF MS − Necessity to build own specialized database of spectral patterns

− Nontrivial processing and analysis of complex data

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

ment of clear morphological hallmarks [33].

+ Feasible, affordable, and robust technique + Intuitive, straight-forward approach + Unbiased and marker-free classification

by other techniques

− Calibration sensitivity

analytical phase

identical morphology [33].

#### *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*

informative peaks, essential for the analysis output [53].

prediction by ANNs. Indeed, well-trained and validated network predicted the number of contaminating cell type with high accuracy in otherwise homogenous and morphologically uniform mixtures. The precision of prediction was dependent on a number of peaks and corresponding peak intensities included in the data matrix. The apparent, dominant peaks with high intensity, did not contribute significantly to the classification. Their exclusion had a negligible effect on the ANN performance, suggesting that there is rather a cumulative effect of minor, but

In the long term cultures of hESCs, abnormal clones with the origin in pristine hESCs are inevitably being generated, selected and, finally, expanded. The CCTL 14 hESC line, which has been propagated over hundreds of passages *in vitro* suffers

*Monitoring of differentiation of hESCs into early lung progenitors (ELEPs) by intact cell MALDI TOF MS. hESCs induced to ELEP differentiation lose gradually the gene signatures associated with pluripotency and acquire the phenotype of lung progenitors. Differentiation trajectory of cells goes through several immature stages and reaches the final, differentiated stage approximately at day 21. Samples for intact cell MALDI TOF MS were collected prior to induction of differentiation (hESCs) at days 1, 3, 8 and 10 (D1, D3, D8, D10) and finally at the final stage (ELEPs). The lung cancer cell line A549 was used as lung-associated, but abnormal sample class. Cluster analysis, such as PCA or a dendrogram, based only on spectral patterns shows clear* 

*separation of classes and indicate that ELEPs represent indeed a distinct cellular entity [66].*

**130**

**Figure 4.**

from chromosomal abnormalities and shows an increased expression of CD30 and CD44 surface molecules, previously shown to be associated with acquisition of the adapted phenotype and karyotype instability [67]. In addition, adapted, high passage hESCs proliferated with reduced doubling time, and showed lower sensitivity to apoptotic stimuli and decreased capability of differentiation, than pristine hESCs. All the low (P29), mid (P72) and high passage (P269) cells maintained high and constant expression of stemness markers, e.g. Sox-2, and also share the identical morphology [33].

We recorded mass spectra from intact P29, P72 and P269 hESCs cultured under identical conditions, processed and assembled into the dataset containing 255 *m/z* values with assigned peak intensities. The mass spectra showed a high level of visual similarity, however the Pearson's correlation analysis revealed disparities in spectral patterns between P29, P72 and P269 cells. Principal component analysis then correctly discriminated P29-pristine and P269-adapted cell to individual distant cluster, while the P72-transition population cluster has been located and scattered between them. Therefore, such a robust dataset of spectral fingerprints recorded from intact hESCs contains sufficient information to distinguish normal and aberrant cells with otherwise identical morphology and expression of stemness markers. Next, we used the same approach to analyze CCTL 14 hESC line stimulated to differentiation. Generally, cell differentiation means acquisition of novel phenotypic properties, and morphological hallmarks, as well as various molecular mechanisms, involved in cell functioning. Retinoic acid (RA) is a potent inducer of embryonic patterning and neurogenesis *in vivo*, so hESCs induced to the differentiation by RA acquire the neuronal phenotype within several weeks. Mass spectra generated from cells treated with RA for only 24 hrs, contained spectral regions which allowed detection of such early differentiation events even prior to development of clear morphological hallmarks [33].

To test a routine, pre-clinical application, we used Intact Cell MALDI TOF MS to monitor the differentiation of hESCs to early lung progenitors (ELEPs). ELEPs, being the direct precursors of lung cells (pneumocytes), represent an important resource for tissue engineering, pollutant testing and pharmacological analyses. ELEPs gradually differentiate from hESCs under specific culture conditions through several stages (D1-D10). Differentiating immature cells lack an unambiguous marker, which would distinguish them from ELEPs. In addition, during differentiation process aberrant cells with unwanted properties may appear in culture. Intact


#### **Table 2.**

*Advantages (+) and limits (*−*) of Intact Cell MALDI TOF MS in quality control of hESCs culture in clinical grade laboratory.*

Cell MALDI TOF MS was able to distinguish individual immature stages from hESCs and from ELEPs, as well as from lung cancer cell line (**Figure 4**).

In summary, Intact Cell MALDI TOF MS coupled with advanced statistics provides an efficient tool for revealing aberrant cells in culture or following differentiation trajectories of pluripotent stem cells and progenitors. The advantages and limits of Intact Cell MALDI TOF MS in quality control of clinical grade stem cell cultures are summarized in **Table 2**.
