The unique potential of human ESCs (hESCs) to differentiate into three main germ layers and subsequent to any cell type of human body brought them into forefront of biomedical research with a caveat of forming teratoma in vivo during differentiation. However, recent progress in cell fate control, directed differentiation, and tissue engineering crossed the boundaries of laboratory and extended to the arena of regenerative medicine. hESC lines are conventionally derived from the inner cell mass (ICM) of preimplantation-stage blastocysts, morula-stage embryos, or late-stage blastocysts and express pluripotency markers (transcription factors—Oct4, Sox2, Nanog; surface antigens—SSEA-4, SSEA-3; proteoglycans—TRA-1-60, TRA-1-81) [1]. To maintain the undifferentiated state, these cells are co-cultured with a support or feeder layer derived from mouse embryonic fibroblast (MEF) that provides all the essential growth factors [1]. The reported success rate for hESC derivation is highly variable, possibly due to variation in embryo quality and culture conditions. Major progress had happened in derivation, propagation, cryopreservation, and efficient passaging of hESCs. Since clinical application of hESCs critically depends on well-characterized growth and differentiation of stem cells, much effort was put in developing conditioned media that will enable feeder-free growth of ESC cells and eliminate animal products [2, 3]. The original culture system for the maintenance of hESC using MEF feeder cell layer support possesses risk of zoonosis transmitted by animal pathogens, potential activation of animal retroviruses, and possibility of immune rejection due to the presence of nonhuman sialic acid. Several approaches such as use of extracellular matrix (ECM) derived from MEF than living feeder cells, hESC-derived fibroblasts, defined culture medium containing components solely derived from purified human material, MEF-conditioned Matrigel layer to establish and maintain clinical-grade hESCs cell lines are in progress [4]. However, these xeno-free and feeder-independent culture systems are costly and laborious and may lead to abnormal karyotype during long-term culture. Recently, Akopian et al. in conjunction with the Internal Stem Cell Initiative Consortium (ISCIC) compared several commercially available ESC culture media with Knockout Serum Replacer, FGF-2, and MEF cell layers for propagation of several hESC cell lines established in five different laboratories and showed that only mTeSR1 and STEMPRO were able to support most cell lines up to 10 passages [2].

The next major challenge for translational application of hESC is to direct their differentiation toward a specific cell lineage. The pioneer study by Itskovtz-eldor et al. [5] showed that hESC cells were capable of forming “embryoid bodies” (EB) comprised of three embryonic germ layers. In this study, hESCs were grown in suspension to induce their differentiation into EBs. Formation of in vitro EB required special cocktail of supplements and growth factors such as glutamine, beta-mercaptoethanol, nonessential amino acids, leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF). Under these conditions, majority of the cells remained in an undifferentiated state. For the formation of EBs, ES cells were transferred using either collagenase or trypsin/EDTA to plastic petri plates to allow aggregation and prevent adherence. About one million ES cells were plated in each of the 50-mm petri plates, and the hEBs were grown in the same culture medium without LIF and bFGF. The differentiation status of the human ES cells and EBs was determined by the expression pattern of several lineage-specific markers such as gamma-globin (hematopoietic cells), alpha-cardiac actin (myocardial cells), neurofilament (neuronal cells), and alpha-fetoprotein (endodermal cells).

Recently, Chen et al. performed a stepwise differentiation of ESCs to insulin-secreting functional beta-cells where ESCs first formed a definitive endoderm in the presence of indolactam-V and FGF, then Pdx1-expressing pancreatic progenitors, followed by the formation of endocrine progenitors and eventually insulin-producing beta-cells (Fig. 2.3) [6, 7].

Thus, strategic development of tissue-specific adult cells with fully functional potential from undifferentiated ES cells is possible and holds great promise for translational application.

Transplantation of hESC-derived cells into human patients:

The first clinical trial using hESC-derived retinal pigment epithelium (RPE) to establish the safety and tolerability in patients suffering from Stargardt’s macular dystrophy and dry age-related macular degeneration was reported by Schwartz et al. [8]. They transplanted a low number of (5 × 10⁴) RPE cells into subretinal space of patient’s eye suffering from different forms of macular degeneration. Preoperative and postoperative ophthalmic examinations such as visual acuity, fluorescein angiography, optical coherence tomography, and visual field testing were performed for its validation (clinicaltrials.gov #NCT01345006 and #NCT01344993). This pilot study generated enthusiasm and hope for cell therapy trials in humans with ESCs, which was sidelined due to the adverse effect of generation of teratoma and ethical regulation. Since isolation of ESCs requires creation, treatment, and destruction of human embryos, hESC research always faces criticism and tight ethical regulation. The effort then moved toward using of adult stem cells, which in spite of limited differential potential has turned out to be a great source for cell therapy application.

Each adult organ in human body harbors a small population of stem cells that have the ability to maintain tissue homeostasis. These “adult stem cells” remain in quiescent or nondividing state until activated by any injury or disease, and they have limited ability to differentiate into organ/tissue-specific lineages. The common ones are HSCs, mesenchymal stem cells (MSCs), neuronal stem cells, umbilical stem cells, cardiac stem cells, retinal stem cells, and limbal stem cells that reside in their respective tissues. Unlike ES cells, adult stem cells do not require a feeder layer or supporting cells for their growth and thus easier to be engineered using different media, growth factors, and small molecules. They also do not pose a risk for developing teratoma and thus preferable in regenerative medicine and stem cell therapy. However, immune rejections of adult stem cells pose serious challenge in certain cases.

While stem cell therapy is emerging as a promising alternative for diseases and genetic disorders where drugs or gene therapy fail, it is limited by availability and stringent culture conditions. Exploiting epigenetic influence on phenotypic outcome, researchers have developed powerful genetic platforms for reversal of differentiated adult cells back to an embryonic state. Such reprogrammed cells are known as “induced pluripotent stem cells (iPSCs),” and the reprogramming strategies include “therapeutic cloning” and “nuclear reprogramming.” Both these strategies act through ectopic introduction of a small number of pluripotency-associated transcription factors into differentiated tissue-specific cells. iPSCs have the ability to differentiate into any of the three germ layers, ectoderm, mesoderm, and endoderm and the respective lineage-specific fully differentiated and functional cells/tissues.

The concept of iPSCs demonstrated long back by Sir John Gurdon when he successfully cloned a frog using intact nuclei from intestinal epithelium cells of [42]. Later, he showed that even nuclei from terminally differentiated adult cells (e.g., blood cells, skeletal muscle, and kidney cells) could generate Xenopus larvae with nuclear transfer [42]. Decades later, in 2006, Takahashi et al. demonstrated the ability of adult mouse fibroblasts to reprogram themselves into pluripotent stem cells by introduction of four key transcription factors (Oct3/4, Sox2, c-Myc, and Klf4) [43]. In November 2007, two independent studies were published simultaneously on successful transformation of differentiated human cells into pluripotent stem cells. While Takahashi et al. used retroviral delivery Oct3/4, Sox2, Klf4, and c-Myc combinations to induce pluripotency in human fibroblasts, Yu et al. delivered Oct4, Nanog, Sox2, and LIN28 by lentiviral transduction in hESC-derived mesenchymal cells to induce pluripotency [44, 45]. These groundbreaking experiments by Sir Gurdon and Yamanaka and his group were acknowledged by Nobel Prize award in 2010.

The most exciting and oversimplified part of iPSC generation is that a combination of only four transcription factors is able to reverse the differentiation process. To identify this main core of pluripotent factors, Yamanaka et al. (2006) evaluated twenty-four candidate genes and Thomson et al. (2007) screened sixteen transcription factors in an assay system in which the induction of the pluripotent state could be detected through the development of resistance to neomycin gene. Both the groups identified Oct4, Sox2, and Nanog as the major pluripotency determinants. Though promising, the current iPSC reprogramming method experiences certain drawbacks such as

Thus, alternative approaches such as use of purified transcription factors, replacement of c-Myc, and strategy to avoid drug resistance selection method are being explored [47].

Their et al. [48] reported generation of TAT-modified cell permeate versions of recombinant Oct4 and Sox2 proteins (Oct4 TAT and Sox2 TAT), and later, Zhou et al. generated protein-piPSCs from murine embryonic fibroblasts. The deleterious effects of c-Myc could be circumvented by using n–myc, and host cell need not be drug resistant if using serum-free condition for iPSCs generation [49]. Recently, miRNA particularly the miR302/367 cluster was used to generate iPSCs from mouse and human somatic cells without adding the exogenous transcription factors. This miRNA-based reprogramming was found to be more efficient (twofold) than the standard Oct4/Sox2/Klf4/Myc-mediated reprogramming and ultimately overcome the deleterious effect of c-Myc reactivation [50].

Using the above-mentioned methods, one can now generate individual-specific iPS cell lines to derive patient-specific progenitor cells and eliminate immune rejection crisis. Moreover, iPSC-based technology will facilitate the production of cell line panels that closely reflect the genetic diversity of a population enabling the discovery, development, and validation of therapies tailored for each individual. Till today, iPSCs has been generated from ten different species mouse, human, rhesus monkey, rat, dog, rabbit, horse, and bird [43, 44, 51–58] and into various lineages as listed in Table 2.2.

However, it still would be a long way for iPSCs to reach the clinic, which requires stringent and systematic validation of lineage-specific differentiation.

For centuries, microscopy is an indispensable tool for visualizing dynamics of biomolecules in live cells. Optical microscopic techniques including conventional light (phase contrast) microscopy, fluorescence microscopy, confocal and multiphoton microscopy, intravital microscopy (IVM) have emerged as powerful tools for noninvasive monitoring and characterization of engineered stem cells and tissues [67].

Though microscopic imaging techniques can generate critical information, they are restricted at cellular level and do not depict the kinetics, distribution, and location of in vivo differentiation of stem cells in a living subject. Even IVM requires an artificially created “window chamber” or “tissue flap” at the body surface and is not applicable for deep tissue imaging. Recent developments in macroscopic or in vivo imaging modalities enable the “visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living systems.” Along with other applications, these modalities are now widely used for imaging stem cell delivery, migration and localization, cell viability, and therapeutic effects in various diseases [86–88].

The Six commonly used modalities for imaging stem cell therapy are ultrasound, CT, SPECT, PET, MRI, and optical. For many instances, multimodality approaches that can collectively assess different parameters specific for each modality are gaining popularity. For imaging the stem cells in vivo, it is required to label the cells with an appropriate probe. The two important methods for labeling cells are as follows:

Direct labeling of cells poses limitation to long-term monitoring since the signal gets diminished by dilution or loss of labeling agents via cell division or differentiation. This limitation can be overcome with “indirect-labeling strategy” where cells are exogenously labeled with reporter genes (bioluminescence, fluorescence, PET, SPECT, or MR reporters) and then implanted for imaging. Indirect labeling requires genetic manipulation of the cells that are often not possible to follow in clinics [86–90]. However, both the strategies have own advantages and disadvantages and should be implemented by experimental need.