From fertilised oocyte to cultivated meat - harnessing bovine embryonic stem cells in the cultivated meat industry.

Global demand for animal protein is on the rise, but many practices common in conventional production are no longer scalable due to environmental impact, public health concerns, and fragility of food systems. For these reasons and more, a pressing need has arisen for sustainable, nutritious, and animal welfare-conscious sources of protein, spurring research dedicated to the production of cultivated meat. Meat mainly consists of muscle, fat, and connective tissue, all of which can be sourced and differentiated from pluripotent stem cells to resemble their nutritional values in muscle tissue. In this paper, we outline the approach that we took to derive bovine embryonic stem cell lines (bESCs) and to characterise them using FACS (fluorescence-activated cell sorting), real-time PCR and immunofluorescence staining. We show their cell growth profile and genetic stability and demonstrate their induced differentiation to mesoderm committed cells. In addition, we discuss our strategy for preparation of master and working cell banks, by which we can expand and grow cells in suspension in quantities suitable for mass production. Consequently, we demonstrate the potential benefits of harnessing bESCs in the production of cultivated meat.

Engineered marble-like bovine fat tissue for cultured meat.

Cultured meat can provide a sustainable and more ethical alternative to conventional meat. Most of the research in this field has been focused on developing muscle tissue, as it is the main component of meat products, while very few studies address cultured fat tissue, an essential component in the human diet and determinant of meat quality, flavor, juiciness, and tenderness. Here, we engineered bovine fat tissue for cultured meat and incorporated it within engineered bovine muscle tissue. Mesenchymal stem cells (MSCs) were derived from bovine adipose tissue and exhibited the typical phenotypic profile of adipose-derived MSCs. MSC adipogenic differentiation and maturation within alginate-based three-dimensional constructs were optimized to yield a fat-rich edible engineered tissue. Subsequently, a marble-like construct, composed of engineered bovine adipose and muscle tissues, was fabricated, mimicking inter- and intra-muscular fat structures.

3D-printable plant protein-enriched scaffolds for cultivated meat development.

Cultivated meat harnesses tissue engineering (TE) concepts to create sustainable, edible muscle tissues, for addressing the rising meat product demands and their global consequences. As 3D-printing is a promising method for creating thick and complex structures, two plant-protein-enriched scaffolding compositions were primarily assessed in our work as 3D-printable platforms for bovine satellite cells (BSC) maturation. Mixtures of pea protein isolate (PPI) and soy protein isolate (SPI) with RGD-modified alginate (Alginate(RGD)) were evaluated as prefabricated mold-based and 3D-printed scaffolds for BSC cultivation, and ultimately, as potential bioinks for cellular printing. Mold-based protein enriched scaffolds exhibited elevated stability and stiffness compared to ones made of Alginate(RGD) alone, while allowing unhindered BSC spreading and maturation. Extrusion based 3D-printing with the two compositions was then developed, while using an edible, removable agar support bath. Successfully fabricated constructs with well-defined geometries supported BSC attachment and differentiation. Finally, cellular bioprinting was demonstrated with PPI-enriched bioinks. Cell recovery post-printing was observed in two cultivation configurations, reaching ∼80-90% viability over time. Moreover, cells could mature within 3D-printed cellular constructs. As animal-derived materials were avoided in our scaffold fabrication process, and pea-protein is known for its low allergic risk, these findings have great promise for further cultivated meat research.

Safety and efficacy of human embryonic stem cell-derived astrocytes following intrathecal transplantation in SOD1G93A and NSG animal models.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease characterized by the loss of MNs in the central nervous system. As MNs die, patients progressively lose their ability to control voluntary movements, become paralyzed and eventually die from respiratory/deglutition failure. Despite the selective MN death in ALS, there is growing evidence that malfunctional astrocytes play a crucial role in disease progression. Thus, transplantation of healthy astrocytes may compensate for the diseased astrocytes. METHODS: We developed a good manufacturing practice-grade protocol for generation of astrocytes from human embryonic stem cells (hESCs). The first stage of our protocol is derivation of astrocyte progenitor cells (APCs) from hESCs. These APCs can be expanded in large quantities and stored frozen as cell banks. Further differentiation of the APCs yields an enriched population of astrocytes with more than 90% GFAP expression (hES-AS). hES-AS were injected intrathecally into hSOD1G93A transgenic mice and rats to evaluate their therapeutic potential. The safety and biodistribution of hES-AS were evaluated in a 9-month study conducted in immunodeficient NSG mice under good laboratory practice conditions. RESULTS: In vitro, hES-AS possess the activities of functional healthy astrocytes, including glutamate uptake, promotion of axon outgrowth and protection of MNs from oxidative stress. A secretome analysis shows that these hES-AS also secrete several inhibitors of metalloproteases as well as a variety of neuroprotective factors (e.g. TIMP-1, TIMP-2, OPN, MIF and Midkine). Intrathecal injections of the hES-AS into transgenic hSOD1G93A mice and rats significantly delayed disease onset and improved motor performance compared to sham-injected animals. A safety study in immunodeficient mice showed that intrathecal transplantation of hES-AS is safe. Transplanted hES-AS attached to the meninges along the neuroaxis and survived for the entire duration of the study without formation of tumors or teratomas. Cell-injected mice gained similar body weight to the sham-injected group and did not exhibit clinical signs that could be related to the treatment. No differences from the vehicle control were observed in hematological parameters or blood chemistry. CONCLUSION: Our findings demonstrate the safety and potential therapeutic benefits of intrathecal injection of hES-AS for the treatment of ALS.

Scalable Expansion of Pluripotent Stem Cells.

Large-scale expansion of pluripotent stem cells (PSC) in a robust, well-defined, and monitored process is essential for production of cell-based therapeutic products. The transition from laboratory-scale protocols to industrial-scale production is one of the first milestones to be achieved in order to use both human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) as the starting material for cellular products. The methods to be developed require adjustment of the culture platforms, optimization of culture parameters, and adaptation of downstream procedures. Optimization of expansion protocols and their scalability has become much easier with the design of bioreactor systems that enable continuous monitoring of culture parameters, continuous media change, and support software for automated control. This chapter highlights the common properties that are required for production of scalable, reproducible, homogeneous, and clinically suitable cell therapy products. We describe the available platforms for large-scale expansion of PSCs and parameters that should be considered when optimizing the expansion protocols in a scalable bioreactor. All the above are detailed in the light of the requirements and challenges of bringing a cell-based therapeutic product to the clinic and ultimately to the market. We discuss some considerations that should be taken into account, such as cost-effectiveness, good manufacturing practice, and regulatory guidelines. Graphical Abstract.

Compact MRI for the detection of teratoma development following intrathecal human embryonic stem cell injection in NOD-SCID mice.

Stem cells are emerging as a promising new treatment modality for a variety of central nervous system disorders. However, their use is hampered by the potential for the development of teratomas and other tumors. Therefore, there is a crucial need for the development of methods for detecting teratomas in preclinical safety studies. The aim of the current study is to assess the ability of a compact Magnetic Resonance Imaging (MRI) system to detect teratoma formation in mice. Five NOD-SCID mice were injected intrathecally with human embryonic stem cells (hESCs), with two mice serving as controls. In vivo MRI was performed on days 25 and 48, and ex vivo MRI was performed after scheduled euthanization (day 55). MRI results were compared to histopathology findings. Two animals injected with hESCs developed hind-limb paresis and paralysis, necessitating premature euthanization. MRI examination revealed abnormal pale areas in the spinal cord and brain, which correlated histopathologically with teratomas. This preliminary study shows the efficacy of compact MRI systems in the detection of small teratomas following intrathecal injection of hESCs in a highly sensitive manner. Although these results should be validated in larger studies, they provide further evidence that the use of MRI in longitudinal studies offers a new monitoring strategy for preclinical testing of stem cell applications.

Derivation, expansion, and characterization of human embryonic stem cell lines from aneuploid embryos.

Human embryonic stem cells (hESCs) are an invaluable cell source to study human embryogenesis and development and for exploring the nature of human diseases. Moreover, hESCs can serve as an unlimited source of cells for cell therapy. The first hESC lines were derived from frozen blastocyst-stage embryos. In the past 12 years, the field evolved and the hESC lines are derived from pre-embryos in various developmental stages using several techniques. In parallel, the wide use of hESCs triggered the development of materials and methods for expansion of the cell lines derived. Here, we describe our method for derivation, expansion, and characterization of hESC lines from pre-embryos that were diagnosed to carry aneuploid cells and were destined to be discarded.

Identification and classification of chromosomal aberrations in human induced pluripotent stem cells.

Because of their somatic cell origin, human induced pluripotent stem cells (HiPSCs) are assumed to carry a normal diploid genome, and adaptive chromosomal aberrations have not been fully evaluated. Here, we analyzed the chromosomal integrity of 66 HiPSC and 38 human embryonic stem cell (HESC) samples from 18 different studies by global gene expression meta-analysis. We report identification of a substantial number of cell lines carrying full and partial chromosomal aberrations, half of which were validated at the DNA level. Several aberrations resulted from culture adaptation, and others are suspected to originate from the parent somatic cell. Our classification revealed a third type of aneuploidy already evident in early passage HiPSCs, suggesting considerable selective pressure during the reprogramming process. The analysis indicated high incidence of chromosome 12 duplications, resulting in significant enrichment for cell cycle-related genes. Such aneuploidy may limit the differentiation capacity and increase the tumorigenicity of HiPSCs.

Variations of X chromosome inactivation occur in early passages of female human embryonic stem cells.

X chromosome inactivation (XCI) is a dosage compensation mechanism essential for embryonic development and cell physiology. Human embryonic stem cells (hESCs) derived from inner cell mass (ICM) of blastocyst stage embryos have been used as a model system to understand XCI initiation and maintenance. Previous studies of undifferentiated female hESCs at intermediate passages have shown three possible states of XCI; 1) cells in a pre-XCI state, 2) cells that already exhibit XCI, or 3) cells that never undergo XCI even upon differentiation. In this study, XCI status was assayed in ten female hESC lines between passage 5 and 15 to determine whether XCI variations occur in early passages of hESCs. Our results show that three different states of XCI already exist in the early passages of hESC. In addition, we observe one cell line with skewed XCI and preferential expression of X-linked genes from the paternal allele, while another cell line exhibits random XCI. Skewed XCI in undifferentiated hESCs may be due to clonal selection in culture instead of non-random XCI in ICM cells. We also found that XIST promoter methylation is correlated with silencing of XIST transcripts in early passages of hESCs, even in the pre-XCI state. In conclusion, XCI variations already take place in early passages of hESCs, which may be a consequence of in vitro culture selection during the derivation process. Nevertheless, we cannot rule out the possibility that XCI variations in hESCs may reflect heterogeneous XCI states in ICM cells that stochastically give rise to hESCs.

Generation of hepatocytes from human embryonic stem cells.

Human embryonic stem cells (HESCs) are pluripotent cells having a self-renewal capacity. These unique characteristics of HESCs allow them to be an unlimited source of cells that was shown to differentiate into many cell types, among them hepatocytes. The creation of hepatocytes in culture will allow us to further understand the mechanisms involved in the embryogenesis of hepatocytes in humans and to study pathologies related to aberrant differentiation of these cells. The resultant hepatocytes may serve as a source of cells for transplantation and as cells for toxicological studies and drug screening. In the past 10 years, since the derivation of HESCs, various protocols for the differentiation of HESCs to hepatic-like cells were published. In this chapter we detail our protocol for differentiating HESCs into hepatic-like cells through embryoid bodies. We further describe the method for the genetic labeling of the hepatic-like cells derived from the HESCs and their isolation by fluorescence-activated cell sorter. We also summarize the published protocols for differentiation of HESCs into hepatic-like cells.

Human embryonic stem cells as models for aneuploid chromosomal syndromes.

Syndromes caused by chromosomal aneuploidies are widely recognized genetic disorders in humans and often lead to spontaneous miscarriage. Preimplantation genetic screening is used to detect chromosomal aneuploidies in early embryos. Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may serve as models for human syndromes caused by aneuploidies. We have established 25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro development. The hESC lines exhibited morphology and expressed markers typical of hESCs. They demonstrated long-term proliferation capacity and pluripotent differentiation. Karyotype analysis revealed that two-third of the cell lines carry a normal euploid karyotype, while one-third remained aneuploid throughout the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X (Turner syndrome). On the basis of the level of single nucleotide polymorphism heterozygosity in the aneuploid chromosomes, we determined whether the aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene expression profiles of the trisomic cell lines suggested that all three chromosomes are actively transcribed. Our analysis allowed us to determine which tissues are most affected by the presence of a third copy of either chromosome 13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic development. The results presented here suggest that aneuploid embryos can serve as an alternative source for either normal euploid or aneuploid hESC lines, which represent an invaluable tool to study developmental aspects of chromosomal abnormalities in humans.

Human embryonic stem cells from aneuploid blastocysts identified by pre-implantation genetic screening.

Human embryonic stem cells are derived from the inner cell mass of pre-implantation embryos. The cells have unlimited proliferation potential and capacity to differentiate into the cells of the three germ layers. Human embryonic stem cells are used to study human embryogenesis and disease modeling and may in the future serve as cells for cell therapy and drug screening. Human embryonic stem cells are usually isolated from surplus normal frozen embryos and were suggested to be isolated from diseased embryos detected by pre-implantation genetic diagnosis. Here we report the isolation of 12 human embryonic stem cell lines and their thorough characterization. The lines were derived from embryos detected to have aneuploidy by pre-implantation genetic screening. Karyotype analysis of these cell lines showed that they are euploid, having 46 chromosomes. Our interpretation is that the euploid cells originated from mosaic embryos, and in vitro selection favored the euploid cells. The undifferentiated cells exhibited long-term proliferation and expressed markers typical for embryonic stem cells such as OCT4, NANOG, and TRA-1-60. The cells manifested pluripotent differentiation both in vivo and in vitro. To further characterize the different lines, we have analyzed their ethnic origin and the family relatedness among them. The above results led us to conclude that the aneuploid mosaic embryos that are destined to be discarded can serve as source for normal euploid human embryonic stem cell lines. These lines represent various ethnic groups; more lines are needed to represent all populations.

Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures.

Induced pluripotent stem cells (iPSCs) outwardly appear to be indistinguishable from embryonic stem cells (ESCs). A study of gene expression profiles of mouse and human ESCs and iPSCs suggests that, while iPSCs are quite similar to their embryonic counterparts, a recurrent gene expression signature appears in iPSCs regardless of their origin or the method by which they were generated. Upon extended culture, hiPSCs adopt a gene expression profile more similar to hESCs; however, they still retain a gene expression signature unique from hESCs that extends to miRNA expression. Genome-wide data suggested that the iPSC signature gene expression differences are due to differential promoter binding by the reprogramming factors. High-resolution array profiling demonstrated that there is no common specific subkaryotypic alteration that is required for reprogramming and that reprogramming does not lead to genomic instability. Together, these data suggest that iPSCs should be considered a unique subtype of pluripotent cell.

Derivation of euploid human embryonic stem cells from aneuploid embryos.

Human embryonic stem cells (HESCs) are pluripotent cells derived from the inner cell mass of preimplantation embryos. In this study, to isolate new lines of HESCs, we used blastocyst-stage embryos diagnosed as aneuploid in preimplantation genetic screening (PGS). During in vitro fertilization treatments, PGS is widely applied to identify chromosomal aneuploidies, especially in cases of advanced maternal age. Embryos that are detected as carrying aneuploidies are destined to be discarded unless donated for research. From 74 fresh PGS-defined aneuploid embryos, we derived seven HESC lines. Most of the embryos were left to hatch spontaneously through the hole created for blastomere biopsy and further treated by immunosurgery. The seven HESC lines exhibited morphology and markers typical of HESCs and the capacity for long-term proliferation. The derived HESC lines manifested pluripotent differentiation potential both in vivo and in vitro. Surprisingly, karyotype analysis of the HESC lines that were derived from these aneuploid embryos showed that the cell lines carry a normal euploid karyotype. We show that the euploidy was not achieved through chromosome duplication. Alternatively, we suggest that the euploid HESC lines originated from mosaic embryos consisting of aneuploid and euploid cells, and in vitro selection occurred to favor euploid cells. We assume that aneuploid HESC lines could be isolated mostly from embryos that are uniform for the aneuploidy. These results led us to conclude that the aneuploid mosaic embryos that are destined to be discarded can serve as an alternative source for normal euploid HESC lines.

The effect of overexpression of Pdx1 and Foxa2 on the differentiation of human embryonic stem cells into pancreatic cells.

Human embryonic stem cells (HESCs) are pluripotent cells that may serve as a source of cells for transplantation medicine and as a tool to study human embryogenesis. Using genetic manipulation methodologies, we have investigated the potential of HESCs to differentiate into the various pancreatic cell types. We initially created various HESCs carrying the enhanced green fluorescent protein (eGFP) reporter gene under the control of either the insulin promoter or the pancreatic and duodenal homeobox factor-1 (Pdx1) promoter. Our analysis revealed that during the differentiation of HESCs into embryoid bodies (EBs), we could detect green fluorescent cells when eGFP is regulated by Pdx1 promoter but not by insulin promoter. To examine whether we can induce differentiation into pancreatic cells, we have established human embryonic stem cell lines that constitutively express either Pdx1 or the endodermal transcription factor Foxa2. Following differentiation into EBs, the constitutive expression of Pdx1 enhanced the differentiation of HESCs toward pancreatic endocrine and exocrine cell types. Thus, we have demonstrated expression of several transcription factors that are downstream of Pdx1 and various molecular markers for the different pancreatic cell types. However, the expression of the insulin gene could be demonstrated only when the cells differentiated in vivo into teratomas. We conclude that although overexpression of Pdx1 enhanced expression of pancreatic enriched genes, induction of insulin expression may require additional signals that are only present in vivo.

Study of hepatocyte differentiation using embryonic stem cells.

The liver has many crucial functions including metabolizing dietary molecules, detoxifying compounds, and storing glycogen. The hepatocytes, comprising most of the liver organ, progressively modify their gene expression profile during the fetal development according to their roles in the different phases of development. Embryonic stem (ES) cells serve as a major tool in understanding liver development. These cells may also serve as a source of hepatic cells for cellular therapy. In this review, we aim to summarize the research that has been performed in the field of hepatocyte differentiation from mouse and human ES cells. We discuss the various methodologies for the differentiation of ES cells towards hepatic cells using either spontaneous or directed differentiation protocols. Although many protocols for differentiating ES cells to hepatic cells have been developed, the analysis of their status is not trivial and can lead to various conclusions. Hence, we discuss the issues of analyzing hepatocytes by means of the specificity of the markers for hepatocytes and the status of the cells as fetal or adult hepatocytes.

Differentiation and isolation of hepatic-like cells from human embryonic stem cells.

Human embryonic stem cells are pluripotent cells that can serve as a cell source for transplantation medicine, and as a tool to study human embryogenesis. We investigate here the potential of human embryonic stem cells to differentiate into hepatic cells. We have characterized the expression level of liver-enriched genes in undifferentiated and differentiated human embryonic stem cells by DNA microarrays. Our analysis revealed a subset of fetal hepatic enriched genes that are expressed in human embryonic stem cells upon differentiation into embryoid bodies. In order to isolate the hepatic-like cells, we introduced a reporter gene regulated by a hepatocyte-specific promoter into human embryonic stem cells. We isolated clones of human embryonic stem cells that express enhanced green fluorescent protein upon in vitro differentiation. Through immunostaining, we showed that most of these cells express albumin, while some cells still express the earlier expressed protein alpha-fetoprotein. Using fluorescence activated cell sorter, we were able to sort out the fluorescent differentiated cells and expand them for a few more weeks. This is the first report to demonstrate the possibility of purifying differentiated derivatives of human embryonic stem cells and culturing them further. Through confocal microscopy, we detected clusters of hepatic-like cells in 20-day-old embryoid bodies and in teratomas. As observed during embryonic development, we showed that in teratomas, the hepatic-like endodermal cells develop next to cardiac mesodermal cells. In order to examine the secreted factors involved in the induction of hepatic differentiation, human embryonic stem cells were grown in the presence of various growth factors, demonstrating the potential involvement of acidic fibroblast growth factor in the differentiation. In conclusion, given certain growth conditions and genetic manipulation, we can now differentiate and isolate hepatic-like cells from human embryonic stem cells.

Differentiation and genetic manipulation of human embryonic stem cells and the analysis of the cardiovascular system.

Human embryonic stem (ES) cells are pluripotent cells derived from blastocyst-stage embryos. The cells are characterized by their self-renewal capability and by their ability to differentiate into a wide range of cell types. In vivo, injection of the human ES cells into immune-deficient mice generates teratomas harboring derivatives of all three embryonic germ layers. In vitro, spontaneous aggregation of human ES cells results in the formation of embryoid bodies (EBs) comprised of differentiated cells from the three embryonic germ layers. Induced differentiation of ES cells into specific subsets of cells may be generated by treatment with several growth factors. Cardiomyocytes and endothelial cells were among the tissue types identified in vitro, and one of the most dramatic examples for the differentiation of human ES cells is the formation of rhythmic contractions of EBs containing pulsing cardiac muscle cells. Cells of the cardiovascular system were characterized by many molecular markers and by their structural and functional properties. The ability to genetically manipulate human ES cells now allows for the purification of specific cell types. Human ES cells have tremendous value as an in vitro model to study embryonic differentiation and as a source of cells for cellular transplantation in various pathologies among them cardiovascular diseases.