Stem Cell Culture and Organ Culture Chapter Notes | Biology for Year 11 PDF Download

Introduction

• Stem cell study is a significant area of biomedical research with potential for basic and translational research, offering solutions for diseases with no effective therapies.
• Stem cells can be used to alleviate suffering from various diseases, with progress already made in clinical applications.
• The field emphasizes rigorous scientific standards and clinical research to reinforce stem cell basics.
• Stem cells are characterized by their ability to reproduce, originating from the zygote formed through fertilization of sperm and oocyte.
• Zygote development involves cell division into two-celled, four-celled, eight-celled stages, eventually differentiating into specialized cells like muscle, skin, liver, cardiovascular, and epithelial cells.
• During differentiation, some cells become mature, while others remain immature, retaining the potential to differentiate into various specialized cell types; these are called stem cells.
• Stem cells possess self-renewal capabilities through mitotic cell division and can differentiate into a wide range of specialized cell types.
• Stem cells generate interest and debate due to their potential in regenerative medicine, with the first stem cells isolated from blood cells.
• Scientists globally are exploring various stem cell types to regenerate tissues or organs, revolutionizing regenerative medicine.
• Stem cells are non-specialized with inherent self-renewal and potency, enabling differentiation into multiple specialized cell types.
• In multicellular organisms, stem cells endure adverse conditions for extended periods.
• In humans, stem cells are found in the umbilical cord, placenta, inner cell mass of early embryos, some fetal tissues, and certain adult organs.
• Stem cells can develop new cells in vitro to replace damaged tissues or organs, study genetic defects, investigate disease causes and treatments, and test new drug molecules.
• Historical milestones include the 1998 derivation of the first human embryonic stem cell (hESC) line, 2006 development of xeno-free culture conditions, and 2007 generation of human induced pluripotent stem cells (hiPSCs).
• In 2012, Dr. Shinya Yamanaka and Sir John Gordon received the Nobel Prize for discovering that mature cells can be reprogrammed to become pluripotent.
• Clinical trials with hESCs began in 2014, hiPSCs in 2009, and advancements like CRISPR/Cas9 gene editing in hESCs occurred in 2017.

Stem Cell Classification

• Stem cells are classified based on their source and differentiation potency.
• Based on source, stem cells are divided into embryonic stem cells (ESCs), fetal stem cells, and adult stem cells.
• Embryonic stem cells, also called early stem cells, are found in the inner cell mass of the blastocyst after approximately five days of development.
• Adult stem cells, or mature stem cells, are present in the umbilical cord, placenta after birth, and mature body tissues.
• Embryonic stem cells are more promising for clinical applications due to their ability to differentiate into any body tissue, unlike adult stem cells.
• Use of embryonic stem cells in humans is limited by technical safety concerns and ethical dilemmas.
• Adult stem cells have no safety or ethical controversies and were once thought to be irreversible but are now known to exhibit plasticity, allowing differentiation into cells of different tissues.
• Based on differentiation potency, stem cells are categorized as totipotent, pluripotent, multipotent, and unipotent.
• Totipotent stem cells have the highest differentiation potential, capable of producing both embryo and extra-embryonic membranes, forming an entire functional organism (e.g., zygote).
• Cells remain totipotent until the eight-cell stage, after which they specialize to form a blastocyst, with the inner cell mass becoming pluripotent.
• Pluripotent stem cells can differentiate into almost all embryonic cell types except extra-embryonic tissues like the placenta, derived from the inner cell mass of the blastocyst.
• Multipotent stem cells differentiate into a closely related family of cells within a specific lineage, such as hematopoietic stem cells forming various blood cells or neural stem cells forming neurons, oligodendrocytes, and astrocytes.
• Unipotent stem cells produce only their own cell type but retain self-renewal properties, found in differentiated tissues like epidermal, muscle, and endothelial cells, making them promising for regenerative medicine.
• Totipotent and pluripotent stem cells are associated with embryonic life, while multipotent and unipotent stem cells are found in adult life, collectively termed adult or somatic stem cells.

Characteristics of Stem Cells

• Adult stem cells have garnered significant attention for their differentiation abilities, particularly their plasticity, which allows tissue-specific stem cells to adopt fates distinct from their tissue of origin.
• Differentiation into a different cell type requires significant molecular rearrangement, dependent on the competence of responding cells and microenvironmental cues.
• Not all bone marrow cells are competent to differentiate into neurons or hepatocytes, and competent cells require an inductive microenvironment to differentiate against their fate.
• Pathways for somatic cells to switch lineages include trans-determination, transdifferentiation, dedifferentiation, heterogeneity, pleiotropy, and fusion.
• Trans-determination involves a stem cell switching to another stem cell type, redirecting its potential to produce different cell lineages.
• Transdifferentiation allows a differentiated cell to adopt the phenotype of another differentiated cell type.
• Dedifferentiation, common in lower vertebrates but rare in mammals, involves adult stem cells reverting to master cells capable of differentiating into other cell types.
• Heterogeneity explains the plastic nature of adult stem cells, with clonal level purity as a gold standard, determined by specific surface markers.
• Pleiotropy occurs when plasticity between cells from the same germ layer is due to clonal level impurity.
• Fusion is an alternative mechanism proposed for adult stem cell plasticity.

Applications of Stem Cells

• Stem cell therapies aim to repair damaged tissues that cannot heal naturally, offering hope for chronic illness patients beyond symptom management.
• Therapies involve transplanting cells to grow new tissue or stimulating existing stem cells to produce new tissue.
• Most stem cell therapeutics are experimental and costly, except for bone marrow transplantation, which is well-established.
• Embryonic and adult stem cells are predicted to treat conditions like muscle damage, cancer, Huntington’s disease, Type 1 diabetes, Parkinson’s disease, cardiac failure, celiac disease, and neurological disorders.
• Further research is needed to understand stem cell behavior post-transplantation and their interaction with diseased microenvironments.
• In neurological diseases like Amyotrophic Lateral Sclerosis (ALS), bone marrow stem cells and induced pluripotent stem cells (iPSCs) are under clinical trials to treat motor neuron loss.
• For spinal cord injuries, adult stem cells are investigated to regenerate nerve cells and stimulate severed nerve fiber growth.
• In eye diseases, transplantation of retinal pigment epithelial cells from embryonic and retinal stem cells has improved vision, offering hope for new therapeutics.
• For wound healing, keratinocyte stem cells from hair follicles are cultured to create skin equivalent to the patient’s, reducing rejection and aiding faster healing of injuries, genetic disorders, and burns with lower inflammation compared to conventional grafts.
• In cardiovascular diseases, stem cell therapies aim to restore heart tissue and blood vessel function, addressing ischemia and muscle injury beyond surgical repairs and medications.
• For autoimmune disorders like Type 1 diabetes, hematopoietic stem cell-derived cells are explored to replace degraded pancreatic beta cells, addressing insulin administration challenges.
• In multiple sclerosis, bone marrow and neural stem cells are investigated to regenerate neurons with proper myelin sheaths, addressing immune-mediated damage.
• For rheumatoid arthritis, stem cells are differentiated into chondrocytes to repair cartilage, offering an alternative to drugs that only reduce pain and inflammation.
• In pharmaceutical industries, stem cells serve as models for drug screening, with cardiomyocytes from human embryonic stem cells used for cardiac disease studies and neuronal cells for neurological disorder research.
• Stem cell research faces challenges like immunological rejection, requiring immunosuppressive treatments that increase infection risk; induced pluripotent stem cells from the recipient’s cells may reduce rejection.
• Embryonic stem cells’ indefinite division may induce tumor growth, and their safety is a concern due to potential microbial infections in recipients.

Organ Culture

• Organ culture involves developing a part or whole organ using tissue culture techniques, preserving anatomical relationships and physiological functions in vitro on artificial media.
• Cell culture lacks the in vivo structural complexity, while organ culture maintains tissue architecture, overcoming limitations of conventional cell cultures.
• Preclinical animal trials have limited success in predicting human responses due to microenvironmental framework deficiencies, addressed by modern approaches like 3D cultures, organs-on-a-chip, and organoids.
• Tissues for organ culture must be handled carefully to avoid damage and transported to the lab quickly, ideally within minutes, to minimize deterioration.
• Organ cultures can be analyzed using immunochemistry, autoradiography, and histology to study tissue characteristics.

Characteristics of Organ Culture

• Organ culture aims to preserve the structural design of the organ or tissue, directing it toward normal development.
• Structural integrity is maintained, but the lack of a vascular system restricts tissue size due to limited diffusion, affecting cell polarity.
• Some proliferation occurs in outer cell layers, with cells unified as a single unit, unlike discrete isolated cells in cell culture.
• Cell-to-cell communication and adhesion are preserved, allowing signal exchange via cell interactions.
• Nutrient and gas exchange are limited due to the absence of a vascular system, with diffusion occurring from the periphery, potentially causing necrosis in the tissue center.
• Organ cultures are exposed to high oxygen concentrations, risking oxygen-induced toxicity.
• Tissues are maintained at a gas-liquid interface to facilitate gas exchange and nutrient access, with anchorage to a solid substrate potentially causing cell outgrowth.
• Using hydrophobic surfaces minimizes outgrowth, and optimal liquid levels maintain spherical geometry, while excessive or insufficient liquid levels cause flattening or compromised gas exchange.
• Hyperbaric oxygen or enhanced pure oxygen content enhances oxygen permeation in organ cultures.

Growth and Differentiation

• Growth refers to an increase in cell numbers, while differentiation involves changes in cell function.
• Differentiated cells may lose proliferation ability, and growth termination can induce differentiation regardless of cell density.
• Most organ cultures do not grow extensively, with proliferation limited to outer cell layers due to structural and shape constraints.
• Organ cultures support cellular communications and differentiation, maintaining an appropriate environment.
• Soluble growth factors are supplied to facilitate differentiation in organ cultures.

Whole Embryo Culture

• Spratt (1950s) studied the effects of metabolic inhibitors on developing embryos in vitro, developing whole embryo culture techniques.
• A suitable medium is prepared and poured onto a watchglass, placed on a moist absorbent cotton wool pad in a Petri dish.
• For chick embryo culture, eggs are incubated at 38°C for 40–42 hours to produce embryos.
• The eggshell is sterilized with 70% ethanol, broken into pieces, and kept in 50 ml of balanced salt solution (BSS).
• The vitelline membrane covering the blastoderm is removed and kept in BSS, with adherent membranes removed using forceps.
• The embryo’s developmental stage is observed under a microscope, then placed on a sterile cotton wool pad in the watchglass and incubated at 37.5°C for further development.

Types of Organ Culture

• Histotypic culture involves proliferating a characterized cell line at high density with suitable soluble factors and extracellular matrix, such as vascular endothelial cells forming capillary tubules in a collagen matrix.
• Cellulose sponges coated with extracellular matrix components like collagen allow cells to infiltrate and form glandular structures.
• Organotypic culture co-cultures cells of different lineages to create tissue-like structures, addressing the limitation of histotypic cultures in evaluating heterologous cell interactions.
• For example, co-culturing fibroblast and epithelial cell clones from the mammary gland enables epithelial cells to differentiate functionally, producing milk proteins in an optimal hormonal environment.
• Organotypic cultures form characteristic structures validated by functional differentiation, such as three-dimensional cords where fibroblasts reorganize and are enveloped by epithelial cells.
• Organoids are self-organized 3D tissue cultures derived from stem cells, made from cells, growth factors, and collagen, with potential to replace diseased organs or deliver genetically altered cells, e.g., artificial livers implanted in rats.

Applications of Organ Culture

• Cultured organs can serve as substitutes for transplantable organs, addressing the decreasing availability of donor organs.
• Organs produced from a patient’s own stem cells allow transplantation without immunosuppressive drugs, reducing rejection risks.
• Organ culture facilitates studying tissue behavior in vitro, understanding biochemical and functional characteristics, and comparing them with in vivo organs.
• It is suitable for studying hormones and their effects, individually or in combination, with the mammary gland of mice being a commonly cultured organ.

Limitations of Organ Culture

• Organ cultures rely heavily on histological techniques rather than biochemical and molecular analyses, limiting reproducibility.
• Preparation of replicates is more challenging than in cell culture, with high variations and low reproducibility due to differences in geometry, handling, sampling, and cell type proportions.
• Organ cultures require fresh organs for each experiment, as they cannot proliferate.
• They are expensive and challenging to prepare, focusing on the behavior of integrated tissues rather than isolated cells.

Future Prospects