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Isolation of HESC Stem Cells and Its Clinical Applications

Updated June 24, 2021
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Isolation of HESC Stem Cells and Its Clinical Applications essay

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Abstract

AHESC(HESC) are pluripotent stem cells that derived from ainner cell mass of ablastocyst. Pluripotent HESCs have a great ability to undergo unlimited self-renewal in culture and to differentiate into all cell types in abody. Ajourney of HESCs research is not that smooth, as it has faced several challenges which are limited to not only tumor formation and immunorejection but also social, ethical, and political aspects. Aisolation of HESCs from ahuman embryo is considered highly objectionable as it requires adestruction of ahuman embryo.

Afterward, some countries lifted aban and allowed afunding in HESCs research, but adamage has already been done on aprogress of research. Under these unfavorable conditions, still some progress was made to isolate, culture, and characterize HESCs using different strategies. In this review, we have summarized various strategies used to successfully isolate, culture, and characterize HESCs. Finally, HESCs hold a great promise for clinical applications with proper strategies to minimize ateratoma formation and immunorejection and better cell transplantation strategies.

Introduction

One of amost promising areas in basic research today involves ause of stem cells. These unique cells have acapability to transform and replenish adifferent tissue types that make up abody, and they also represent afundamental building blocks of human development. In general, stem cells can be divided into two broad categories: adult (somatic) stem cells and embryonic stem (ES) cells. Arecent derivation of HESC lines from human blastocyst and human embryonic germ (EG) cell lines from primordial germ cells has aroused intense public and scientific discussion.

This interest stems in part from acontroversy surrounding aorigin of these lines but, more importantly, from awidespread conviction that their availability will have a major impact on several scientific areas. Afocus of apresent review will be to describe aisolation and unique properties of human ES cells. Particular emphasis will be placed on describing aclinical applications of this new technology in avarious fields. These applications will be discussed in acontext of aextensive experience gathered over ayears.

Stem Cells

Stem cells are primitive cells with acapacity to divide and give rise to more identical stem cells or to specialize and form special cells of somatic tissues. Broadly speaking, two types of stem cell can be distinguished: embryonic stem (ES) cells which can only be derived from pre-implantation embryos and have a proven ability to form cells of all tissues of aadult organism (termed `pluripotent’), and `adult’ stem cells, which are found in a variety of tissues in afetus and after birth and are, under normal conditions, more specialized (`multipotent’) with an important function in tissue replacement and repair.

Human Embryonic Stem Cells

Embryonic stem cells (ES cells or ESCs) are pluripotent stem cells derived from ainner cell mass of a blastocyst, an early-stage pre-implantation embryo. Human embryos reach ablastocyst stage 4–5 days of post fertilization, at that time they consist of 50–150 cells. Researchers are currently focusing heavily on atherapeutic potential of embryonic stem cells, with clinical use being agoal for many labs. Potential uses include atreatment of diabetes and heart disease. Acells are being studied to be used as clinical therapies, models of genetic disorders, and cellular/DNA repair. However, adverse effects in aresearch and clinical processes such as tumors and unwanted immune responses have also been reported.

Derivation of Embryonic stem cells

Aterm ES cells originated from aisolation in 1981 of pluripotent stem cell cultures from mouse blastocyst by Evans and Kaufman. Aorigin of human ES cells, in contrast to EC and EG cells is from apreimplantation embryo. These cell lines were derived from aICM cells of human blastocyst, produced by in vitro fertilization for clinical purposes and donated by individuals after informed consent.

AHESClines were created in a manner similar to that of mouse and rhesus16 ES cells. In this process, aouter trophectoderm layer of ablastocyst was selectively removed using specific antibodies (immunosurgery), and aICM cells were isolated and plated on a mitotically inactivated mouse embryonic fibroblast (MEF) feeder layer. Ahuman ES cells were demonstrated to fulfill all acriteria defining embryonic stem cells, namely, derivation from apreimplantation embryo, prolonged undifferentiated proliferation in culture under special conditions, and acapacity to form derivatives of all three germ layers.

To derive human ES cells, cleavage-stage embryos are grown into blastocyst and ainner cell mass is removed. Aembryos from which ainitial HESC lines were derived were produced by in vitro fertilization and donated with ainformed consent of aparents (Figure 1). Ause of such ‘spare’ embryos is widely held to be acceptable; they are created for reproductive purposes for which they are no longer required and it is considered to be ethically superior to use them for medical research rather than destroying them or storing them indefinitely.

Another approach to deriving HESC is somatic nuclear transfer or cloning. This procedure uses atransfer of a somatic cell nucleus from an individual into an enucleated oocyte. Acells are then allowed to undergo embryonic development to ablastocyst stage prior to aisolation from ainner cell mass of ES cells that would be genetically matched to atissues of adonor of anucleus. Agroup that claimed to clone human embryos has also recently reported that they have derived stem cells parthenogenetically from non-human primates. They suggest that their approach provides an alternative to somatic cell nuclear transfer as a means to generate autologous human embryonic stem cells.

Several other methods were also developed to isolate inner cell mass (ICM) from a single human embryo, which include mechanical dissection, where ICM is isolated by mechanical pressure.

AICM can also be isolated by using laser dissection and by using immunosurgery procedures, there are various benefits of using an immunosurgery procedure to isolate ICM, but this also carries some disadvantages. For example, aimmunosurgery procedure requires aculture media which contain guinea pig serum; hence, ause of animal serum makes aimmunosurgery technique not suitable for ageneration of clinical-grade

HESC line. In another method, HESC lines can be isolated from ICM by micro dissection of human blastocyst using fine needles. Laser-assisted biopsy is also amost promising technique for xenon-free isolation of aICM. After ICM isolation, astems cells are grown to generate aESCs using feeder layers.

Difference between mouse and human embryonic stem cells

While adefining characteristics of embryonic stem cells derived from mice and humans are shared, a number of differences exist between atwo cell types. When grown on feeders, HESCs form thinner colonies (2-4 cells thick) than mouse embryonic stem cells (MES) (4-10 cells thick) and show a substantially longer doubling time (36-45 hours for HESCs compared to 12 hours for MES cells).

In vitro Differentiation

One of autmost characteristics of HESCs is to differentiate into all three lineages such ectoderm, mesoderm, and endoderm. As HESCs are pluripotent stem cells, they have unique capabilities to differentiate into all kinds of body cells; for example, HESCs can be differentiated into neurons, cardiomyocyte, and hematopoietic, pancreatic, endothelial etc.

Ectoderm Differentiation

Ectoderm differentiation of HESC can be achieved either by addition of growth factors that induce differentiation to cells expressing neuronal, adrenal or skin related genes, or by allowing spontaneous differentiation. Extensive work has been dedicated to neuronal differentiation, since derivation of various specialized neuronal cells is relatively easy, compared with other cell lineages, and could be of great value for atreatment of neurodegenerative disorders

Neuronal Differentiation

In vitro, HESC derived neurons synthesize neurotransmitters, respond to neurotransmitters and are electrically active. Additionally, a neural-tube like structure is formed when HESC are induced to differentiate into neuronal progenitors. In vivo, when HESC-derived progenitor neurons were transplanted into a newborn rodent brain, they integrated into different locations in abrain.

Mesoderm Differentiation

HESC differentiation into mesoderm lineage cell derivatives is demonstrated by expression of markers specific to cardiomyocytes, endothelial, bone, kidney, urogenital, muscle, and blood cells.

Cardiomyocyte Differentiation

Pulsating HEB are afirst indication of apresence of functional cardiomyocytes derived from HESC. Analysis of acontracting areas in aHEB indicates that they have physiologic characteristics similar to early cardiac tissue. Additionally, progressive stages of early cardiomyocyte differentiation are observed during HEB differentiation. It is also possible to obtain a variety of functional cardiac cells in vitro

Endothelial Differentiation

Endothelial cells were shown to appear during aspontaneous differentiation of HESC. Apresence of endothelial cells within HEB is demonstrated not only by aexpression of endothelial specific markers, but also by asuccessive morphologic progression from endothelial cell clusters to vascular-like structures and to a network like organization. Additionally, isolation of endothelial cells from HEB yields vascular tubes both in vitro and in vivo.

Hematopoietic Differentiation

Two differentiation methods were used to achieve hematopoietic differentiation of HESC: culturing HESC in apresence of stromal cells and aadministration of growth factors such as stem cell factor

(SCF), Fms-like tyrosine kinase 3 ligand (Flt3L), IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), and BMP-4 to HEB. These methods give rise to several types of hematopoietic lineages, including erythroid, myeloid, and lymphoid. In contrast to HESC, hematopoietic stem cells (HSC) demonstrate a limited proliferative capacity. Thus, aability to derive hematopoietic cells from HESC may offer a potential replacement for acurrently used HSC for cell-based therapy.

Osteogenic Differentiation

Recently, several protocols for bone differentiation were published. It was shown that addition of osteogenic supplements to HEB culture results in aexpression of osteogenic markers, mineralization of aculture and aappearance of a principle component of bone matrix. In another study, HESC were shown to respond in a similar way to a mouse ES cells osteogenic differentiation protocol, especially to aaddition of dexamethasone to aculture. Aresulting differentiated cells were transplanted into severe combined immunodeficient mice on a scaffold and demonstrated acapacity to form mineralized tissue in vivo.

Endoderm Differentiation

Isolation and characterization of endoderm derivatives from HESC is of great interest inasmuch as these cells might serve as a source of cells for cell therapy in different pancreatic disorders and liver failure diseases. Recently, differentiation of

HESC to definitive endoderm has been reported. Moreover, there are several reports of specific differentiation to hepatic- and pancreatic-like cells

Hepatic Differentiation

It has been suggested that culturing HESC with sodium butyrate results in cells with an epithelial morphology that express hepatic markers and possess metabolic activity similar to primary adult human hepatocytes. Furthermore, gene expression analysis of HESC throughout differentiation indicates that there are several hepaticassociated genes such as albumin, fibrinogen, and apolipoprotein, which are induced upon differentiation. These hepatic like cells can be successfully isolated by introduction of a reporter gene regulated by a hepatic specific promoter. These cells express hepatic specific markers and are observed adjacent to cardiac mesodermal cells in teratomas, as observed in normal embryonic development.

Pancreatic Differentiation

Pancreatic development is regulated by several factors such as Foxa2 and Pdx1 and by epithelial-mesenchymal interactions. Yet, ainitial steps in normal pancreatic development are still unknown. HESC have been shown to express pancreatic specific markers. However, so far, ageneration of functional beta-like cells has been unsuccessful in that adifferentiated cells failed to secrete insulin when stimulated with glucose, as expected from functional beta cells.

Limitations of HESC in Vitro Differentiation

There are certain cell types that could not be obtained by in vitro differentiation of HESC. A prominent example for this is ainability to obtain insulin secreting pancreatic-cells in culture. Furthermore, some of adifferentiated cell products derived from HESC do not express afull repertoire of markers that characterize there in vivo counterparts, as is illustrated in acase of aHES hepatic like derived cells, in which adult liver transcripts are undetectable. Finally, it is unclear whether all adifferentiated derivatives are indeed functional, an issue that could be addressed only by in vivo transplantation experiments, which are limited to animal models for obvious reasons.

Clinical Applications

As HESCs hold a lot of promises for apatients who are suffering from degenerative diseases, various attempts have been made to explore atherapeutic potentials in humans. Amain objective of stem cell-based therapy is to restore or repair alost or damaged body cells or tissues. Because of aoutstanding potential demonstrated by mouse ES lines, aintroduction of HESC cell lines may hold important research and clinical applications. Specifically, important topics related to cardiac research may include afollowing:

  • investigation of amechanisms involved in early human cardiac lineage commitment, differentiation, and maturation;
  • derivation of a unique in vitro model to study human cardiac tissue;
  • functional genomics, drug and growth factor discovery, drug testing, and reproductive toxicology; and
  • development of cell-based therapies and tissue-engineering strategies.

Other than cardiac related diseases, stem cells also used to treat various diseases like Parkinson’s disease, Alzheimer, diabetes and cancer.

Conclusion

Arecent derivation of HESC has generated much interest, mainly due to their therapeutic potential. HESC have great therapeutic potentials for atreatment of various diseases such as cancer, Parkinson’s disease, Alzheimer’s disease, and diabetes. HESC provide a new approach to astudy human embryo development, which has so far been limited by ainadequacy of available models. In addition, acells may be used as an in vitro system for not only study of adifferentiation of specific cell types, but also evaluation of aeffects of new drugs and aidentification of genes as potential therapeutic targets. Finally, HESC still hold a great promise for atreatment of various degenerative diseases as well as diagnostic applications.

Isolation of HESC Stem Cells and Its Clinical Applications essay

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Isolation of HESC Stem Cells and Its Clinical Applications. (2021, Jun 24). Retrieved from https://samploon.com/isolation-of-hesc-stem-cells-and-its-clinical-applications/

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