**2. Embryonic stem cells and the need of quality control**

Shortly after fertilization, early human embryo is composed of blastomeres equal undifferentiated cells, which have an unlimited potential to develop into any type of embryonic as well as extraembryonic tissue. When the embryo reaches the 8-cell stage, its blastomeres start forming intercellular junctions, maximizing the contact with each other, in a process called compaction. Already in the 16-cell formation, called morula, cells with no contact with the outer environment - the inner cell mass (ICM) - can be recognized. Prior to implantation, morula develops a small cavity, becoming a blastocyst, in which ICM further proliferates and forms embryoblast. Cells of embryoblast, still being pluripotent, differentiate rapidly and build bilaminar germ disc with the distinct layers - hypoblast and epiblast. Epiblast cells represent the essential developmental source for principal embryonic germ layers - ectoderm, mesoderm and endoderm [1].

Mammalian ESCs are derived from undifferentiated ICM of early preimplantation blastocyst. While the embryoblast represents an ephemeral stage in embryonic development, the ESCs, when transferred into long term *in vitro* culture share some characteristics with an immortalized cell line. Specific conditions of the *in vitro* culture prevent differentiation of the ESCs into the embryonic structures, and allow ESCs to keep the molecular machinery necessary for the maintenance of pluripotency and unlimited cell divisions [2].

Development of differentiated structures from embryonic precursor cells is a tightly regulated process. Pluripotent cells, which have not differentiated properly in their developmental time window, or persist ectopically, can form peculiar tumors – teratomas. Teratomas contain regions with haphazard accumulation of mature tissues, such as hair, cartilage, bone, or teeth. Despite the inherent abnormality of teratomas, they were a unique model, which contributed significantly to understanding of embryonic pluripotency and differentiation principles. Indeed, the first cell lines that harbored traits of stem cells were derived from mouse testicular teratocarcinomas [3]. These cells share many features with primordial germ cells, thus they, at the time, served as the first principle model of cell plasticity and embryonic differentiation. Genuine embryonic stem cells were derived from 3.5 dpc mouse blastocyst in 1981 [4], and the long-term culture conditions preventing differentiation were adopted from teratocarcinomas culture protocols [5]. Later, soluble factors critical for maintaining the pluripotency of mouse ESCs (mESCs), e.g. mLIF – mouse leukemia inhibitory factor, and BMP4 – bone morphogenetic factor 4, were identified and allowed mESCs propagation [6, 7].

**123**

**Figure 1.**

*spotted on the target plate of the MS instrument.*

*Intact Cell Mass Spectrometry for Embryonic Stem Cell Biotyping*

The embryonic development of primates including humans, however, differs from embryonic development of rodents. That is why the derivation of human embryonic stem cells has not been achieved until seventeen years later by James Thompson [8]. Currently, hundreds of hESC lines and their derivatives are available through curated stem cell repositories and biobanks [9]. A breakthrough in stem cell research came in 2006 when genetic regulators essential for induction of pluripotency were discovered by Shinya Yamanaka [10]. These transcription factors can reprogram fully differentiated somatic cells, so they obtain undifferentiated stem cell-like phenotype. Such human induced pluripotent stem cells (hiPSCs) provide a genuine stem cell model with no ethical burden associated with hESCs [11]. hiPSCs essentially complement the portfolio of cellular models for description of crucial molecular events during embryogenesis, tissue differentiation and cancer development. Nowadays, ESCs derived from embryoblasts and hiPSCs derived by reprogramming of somatic cells represent an important biological model and hold the promise to various clinical applications, including regenerative medicine, tailor-

The state of pluripotency is developmentally unstable and requires specific molecular machinery to maintain the stem cell phenotype [14, 15]. Developmental trajectories during normal embryogenesis are determined soon, so the complex differentiated structures evolve even in a very early embryo. Sophisticated microenvironment of *in vitro* cell cultures, which is designed and optimized to stimulate unlimited propagation of stem cells and preservation of their full differentiation capability [16], is different from the conditions within the developing embryo. Protocols which represent the golden standard involve additional "feeder" cell layer,

Mouse embryonic fibroblasts (MEFs), human preputial fibroblasts, oviduct lining or fetal smooth muscle cells are commonly used as a feeder layer. Feeder cells provide necessary intercellular contacts and produce extracellular matrix (ECM) along with soluble factors, in order to recreate, to certain extent, the microenvironment of a blastocyst [17]. However, co-culture of hESCs with feeder cells bears the risks of carry-over contamination and induction of the immune reaction in a patient

Culture protocols with animal additives are therefore not suitable for the direct use in humans. hESCs culture on defined surfaces coated with ECM mixtures or pure protein layers in complex medium supplemented with essential cytokines

*(A) Single colony of hESCs co-cultured with feeder fibroblast layer (MEFs) as visualized by light microscopy. (B) Scanning electron micrograph of intact hESCs mixed with matrix containing acidified sinapinic acid and* 

being exposed to antigens derived from animal feeder layer upon grafting.

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

made cell therapy and drug testing [12, 13].

which supports cultured stem cells (**Figure 1A**).

#### *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. Embryonic stem cells and the need of quality control**

Shortly after fertilization, early human embryo is composed of blastomeres equal undifferentiated cells, which have an unlimited potential to develop into any type of embryonic as well as extraembryonic tissue. When the embryo reaches the 8-cell stage, its blastomeres start forming intercellular junctions, maximizing the contact with each other, in a process called compaction. Already in the 16-cell formation, called morula, cells with no contact with the outer environment - the inner cell mass (ICM) - can be recognized. Prior to implantation, morula develops a small cavity, becoming a blastocyst, in which ICM further proliferates and forms embryoblast. Cells of embryoblast, still being pluripotent, differentiate rapidly and build bilaminar germ disc with the distinct layers - hypoblast and epiblast. Epiblast cells represent the essential developmental source for principal embryonic germ layers - ectoderm,

Mammalian ESCs are derived from undifferentiated ICM of early preimplantation blastocyst. While the embryoblast represents an ephemeral stage in embryonic development, the ESCs, when transferred into long term *in vitro* culture share some characteristics with an immortalized cell line. Specific conditions of the *in vitro* culture prevent differentiation of the ESCs into the embryonic structures, and allow ESCs to keep the molecular machinery necessary for the maintenance of pluripo-

Development of differentiated structures from embryonic precursor cells is a tightly regulated process. Pluripotent cells, which have not differentiated properly in their developmental time window, or persist ectopically, can form peculiar tumors – teratomas. Teratomas contain regions with haphazard accumulation of mature tissues, such as hair, cartilage, bone, or teeth. Despite the inherent abnormality of teratomas, they were a unique model, which contributed significantly to understanding of embryonic pluripotency and differentiation principles. Indeed, the first cell lines that harbored traits of stem cells were derived from mouse testicular teratocarcinomas [3]. These cells share many features with primordial germ cells, thus they, at the time, served as the first principle model of cell plasticity and embryonic differentiation. Genuine embryonic stem cells were derived from 3.5 dpc mouse blastocyst in 1981 [4], and the long-term culture conditions preventing differentiation were adopted from teratocarcinomas culture protocols [5]. Later, soluble factors critical for maintaining the pluripotency of mouse ESCs (mESCs), e.g. mLIF – mouse leukemia inhibitory factor, and BMP4 – bone morphogenetic

factor 4, were identified and allowed mESCs propagation [6, 7].

identification.

mesoderm and endoderm [1].

tency and unlimited cell divisions [2].

The beginnings in Mass Spectrometry (MS) development in biology correspond with the discovery of soft ionization techniques, such as Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption-Ionization (MALDI) by John Fenn (1988) and Koichi Tanaka (1988), respectively, who have been awarded with the Nobel Prize in chemistry in 2002 for these findings. MALDI MS coupled with Time-Of-Flight (TOF) analysis has been then successfully introduced to many fields beyond analytical chemistry, including proteomics, metabolomics, clinical microbiology and structural biology. MALDI TOF MS allows precise identification, structural analysis and quantification of various, predominantly soluble, analytes. MALDI TOF MS can generate molecular or spectral patterns that can be assigned to specific cell types or states, when intact cells are used as the analyte. Moreover it provides sufficient discrimination capacity due to a high variability in molecular species, constituting the whole spectrum a biomarker even without preceding peak

**122**

The embryonic development of primates including humans, however, differs from embryonic development of rodents. That is why the derivation of human embryonic stem cells has not been achieved until seventeen years later by James Thompson [8]. Currently, hundreds of hESC lines and their derivatives are available through curated stem cell repositories and biobanks [9]. A breakthrough in stem cell research came in 2006 when genetic regulators essential for induction of pluripotency were discovered by Shinya Yamanaka [10]. These transcription factors can reprogram fully differentiated somatic cells, so they obtain undifferentiated stem cell-like phenotype. Such human induced pluripotent stem cells (hiPSCs) provide a genuine stem cell model with no ethical burden associated with hESCs [11]. hiPSCs essentially complement the portfolio of cellular models for description of crucial molecular events during embryogenesis, tissue differentiation and cancer development. Nowadays, ESCs derived from embryoblasts and hiPSCs derived by reprogramming of somatic cells represent an important biological model and hold the promise to various clinical applications, including regenerative medicine, tailormade cell therapy and drug testing [12, 13].

The state of pluripotency is developmentally unstable and requires specific molecular machinery to maintain the stem cell phenotype [14, 15]. Developmental trajectories during normal embryogenesis are determined soon, so the complex differentiated structures evolve even in a very early embryo. Sophisticated microenvironment of *in vitro* cell cultures, which is designed and optimized to stimulate unlimited propagation of stem cells and preservation of their full differentiation capability [16], is different from the conditions within the developing embryo. Protocols which represent the golden standard involve additional "feeder" cell layer, which supports cultured stem cells (**Figure 1A**).

Mouse embryonic fibroblasts (MEFs), human preputial fibroblasts, oviduct lining or fetal smooth muscle cells are commonly used as a feeder layer. Feeder cells provide necessary intercellular contacts and produce extracellular matrix (ECM) along with soluble factors, in order to recreate, to certain extent, the microenvironment of a blastocyst [17]. However, co-culture of hESCs with feeder cells bears the risks of carry-over contamination and induction of the immune reaction in a patient being exposed to antigens derived from animal feeder layer upon grafting.

Culture protocols with animal additives are therefore not suitable for the direct use in humans. hESCs culture on defined surfaces coated with ECM mixtures or pure protein layers in complex medium supplemented with essential cytokines

#### **Figure 1.**

*(A) Single colony of hESCs co-cultured with feeder fibroblast layer (MEFs) as visualized by light microscopy. (B) Scanning electron micrograph of intact hESCs mixed with matrix containing acidified sinapinic acid and spotted on the target plate of the MS instrument.*

(e.g. bFGF, activin) may be a solution, if combined with rigorous quality control of other culture parameters (**Table 1**) [18, 19].
