Why Stem Cells?

Why Stem Cells?

by | Feb 18, 2021 | Uncategorized

Within the body, the majority of cells are destined to convey specific functions e.g., erythroid blood cells deliver oxygen, endothelial cells form vasculature, neurons mediate communication by conducting electric impulses. Stem cells are unspecified cells that possess the ability to produce an exact copy of itself, self-renew, and to give rise to lineage-committed progenies producing many types of functional cells in the body. The unique and important function of stem cells is to build and sustain multicellular organisms through their lifespan.

Stem cells can be categorized by their differential potential into totipotent, pluripotent, multipotent, oligopotent, and unipotent. Totipotent stem cells exist at a very early stage of the development and give rise to both embryonic and extra-embryonic tissues such as placenta.  Embryonic stem cells are pluripotent as they can give rise to tissues of three germ layers: ectoderm, mesoderm, and endoderm. The regeneration potential of multipotent stem cells comprises a variety cell types but is restricted to one of three lineages. Oligopotent stem cells can only differentiate into a few cell types such as lymphoid or myeloid stem cells, whereas unipotent cells can produce only one cell type. Totipotent and embryonic stem cells exist only at the early stage of development, whereas multipotent, oligo- and unipotent stem cells are found in many adult tissues and organs e.g., bone marrow, adipose tissue, brain, and blood. Hematopoietic, mesenchymal, and endothelial stem cells found in the placenta, and umbilical cord are considered fetal stem cells.

The stem cell’s ability to self-renew is of upmost necessity to ensure the maintenance of the stem cell population and their regenerative functions. Self-renewal is achieved through symmetric or asymmetric cell division. In the symmetric stem cell division, two daughter cells are identical to each other and to the parental cell. Asymmetric cell division occurs when only one of two progenies is identical to the parental cell and another daughter cell has slightly different molecular features. This bias triggers a further commitment and generation of lineage-specific progenitors that ultimately differentiate into fully functional cells replacing old and damaged tissues.

The proliferative, differentiation, and regenerative capacity of stem cells can provide many advances in areas of medicine, research, and drug discovery. Currently, umbilical cord blood- or adult bone marrow-derived hematopoietic stem cells’ transplantation is the most common type of stem cell therapy, successfully applied for treatments of inherited blood disorders and leukemia. Pluripotent stem cells generated from patient-derived tissues with inherited or acquired genetic aberrations allow for more precise and personalized approaches to therapeutics’ design. Pluripotent and other models of cancer help to uncover tumor evolution, mechanisms of drug resistance, and cancer stem cell self-renewal.  

Generation of red blood cells from human pluripotent stem cells.

Generation of red blood cells from human pluripotent stem cells.

by | Mar 7, 2017 | Hematopoiesis

Erythrocytes or red blood cells (RBCs) are the most abundant cell population comprising of ~45% of the total blood volume, whose main function is to deliver oxygen to the body tissues. Red blood cells lack a nucleus and most organelles to provide maximum space for hemoglobin – a complex metalloprotein containing heme groups whose iron atoms temporarily bind to oxygen molecules and release them throughout the body.  Mammalian erythroid progenitors originate from a megakaryocyte-erythroid progenitor (MEP) and undergo the gradual process toward terminal differentiation. Two globin gene switches occur during development: the embryonic to fetal globin switch, which coincides with the transition from embryonic (yolk sac) to definitive (fetal liver) hematopoiesis; and fetal to adult switch, which occurs during the perinatal period. During erythroblast differentiation, the chromatin condenses while the hemoglobin concentration increases. Chromatin condensation involves histone deacetylation and unknown signals that activate the Rac-GTPases-mDia2 pathway, which is required for the formation of a contractile actin ring and subsequent enucleation, the process in which the nucleus is rapidly squeezed out of the cell. Erythroid precursors proliferate, differentiate, and enucleate within specialized niches called erythroblastic islands. These hematopoietic compartments are composed of erythroblasts surrounding a central macrophage. The central macrophage communicates with erythroblasts through a number of signaling molecules and phagocytizes their nuclei after enucleation.

In 1977, the American biochemist Eugene Goldwasser isolated the human protein erythropoietin (EPO), which supports the generation of mature blood cells. EPO became a blockbuster product that changed the lives of millions of patients suffering from hematological diseases. Although EPO with other specific additives allow red blood cells to mature in vitro without a supportive role of macrophages, the resulting proliferation and enucleation efficiency of red blood cells is lower than their capacities in vivo, suggesting the importance of a niche microenvironment. Blood transfusions are a common treatment for severe anemia and massive blood loss due to trauma. A type O-negative red blood cell can be transfused to patients of all blood types and is always in great demand. Thus, the derivation of (O)Rh-negative RBCs from PSCs could be an effective way to overcome shortages in donated red blood cells. Red blood cells can be produced from human pluripotent stem cells (hESCs and iPSCs) through various differentiation systems such as an embryoid body (EB) formation and coculturing hPSCs on top of stromal feeder cells.

 

In general, the existing methods are sufficient for a large-scale production of hPSC-derived red blood cells, whose in vitro expansion capacity is greater than the expansion potential of the bone marrow, peripheral blood, or even cord blood-derived erythroid progenitors. Despite the large amounts of RBCs were obtained in many studies, the majority of the resulting RBCs expresses embryonic ε– and fetal γ-globins with low levels of detectable adult β-globin. Although no differences were observed between hiPSC and hESC lines in terms of erythroid commitment and expression of erythroid markers, iPSC-derived red blood cells have lower proliferation activity and produce less enucleated cells.Robert Lanza’s group suggested the idea of developing an early hemato-endothelial progenitor, a hemangioblast, which can be expanded and cryopreserved. This study, published in Nature Methods in 2007, demonstrated the regenerative properties of blast cells that differentiate into multiple hematopoietic lineages as well as into endothelial cells. The extended coculture of these cells on OP9 feeders facilitated enucleation in up to 65% of cells and the expression of β-globin in up to 15% of the cells.

 

In 2010 a group of French scientists announced the complete differentiation of human induced pluripotent stem cells into definitive erythrocytes. Lapillonne and colleagues employed a feeder free, two-step differentiating system to produce mature blood cells from hESCs and  iPSCs. In the first step, researchers initiated blood development by conditioning embryoid bodies in the presence of cytokines. To obtain mature erythrocytes, they further cultured cells in the presence of EPO, SCF, IL3 and 10% of human plasma for another 25 days. The resulting population contained up to 10% of enucleated cultured RBC from hiPSC, and 66% of enucleated cultured RBC from H1-hESC. The vast majority (~93%) of PSCs-derived red blood cells expressed the tetrameric form of fetal hemoglobin HbF (α2γ2). The CO-rebinding kinetics of hemoglobin from hESC- and hiPSC-derived erythroid cells was almost identical to those of cord blood suggesting that the HbF in these erythroid cells is functional.

Several studies have shown a time-dependent increase in β-globin expression, the oxygen dissociation curve and G6PD activities similar to normal RBCs. Nevertheless, significant progress is needed in the production of terminally differentiated /enucleated erythrocytes. Thus, at least two major steps are required for future therapeutic use of in vitro generated RBCs: (i) finding a cost-effective method for generating fully maturated, enucleated erythrocytes, and (ii) the evaluation of their biophysical parameters such as membrane surface potential, pliability, half-life in vivo, hemoglobin packing, gas exchange properties, and immunogenicity.

From pluripotency to blood.

From pluripotency to blood.

by | Mar 7, 2017 | Hematopoiesis

Hematopoietic differentiation of pluripotent stem cells in vitro

Pluripotent stem cells (PSCs) derived from the inner part of a blastocyst (embryonic stem cells, ESCs) or through reprogramming of terminally differentiated adult cells (induced pluripotent stem cells, iPSCs) are capable of self-renewal and differentiation into almost all cell types in the human body. Their differentiation capacities and proliferation potential make pluripotent stem cells a promising source of cells for various clinical applications including regenerative medicine. Blood is considered to be a connective tissue both functionally and embryologically. It originates from the mesodermal layer, the same germ layer that give rise to the other connective tissues such as bone, cartilage and muscle. Blood cells and blood vessels develop in a parallel and form a functional circulating system. Various studies have shown, that hematopoietic differentiation of PSCs in vitro closely resembles early steps of blood development in the embryo and induces blood forming cell populations with mesodermal and hemato-endothelial properties. Different types of mature blood cells were successfully generated from murine, primate and human pluripotent stem cells. Here, we will briefly review the major in vitro systems of hematopoietic differentiation from PSCs.

Embryoid Body formation

Hematopoietic differentiation of PSCs can be carried out in either a two-dimensional system (2D), where cells are attached to the plate during differentiation, or in a three-dimensional system (3D), where isolated cells are dispersed into a liquid or a semisolid medium to form embryoid bodies (EBs). Embryoid bodies (EBs) are spherical structures that are formed by pluripotent stem cells grown in non-adherent culture conditions (3D system).

Differentiation of PSC in aggregates mimics three-dimensional embryonic development and yields the establishment of cell adhesion, paracrine signaling and a microenvironment similar to native tissue structures. Thus, EB formation is often used as a method for initiating spontaneous differentiation of PSC towards all three germ lines. Differentiation in the presence of growth factors specific for mesoderm (BMP4, FGF, activin A) and blood formation (VEGF, SCF, Flts3, IL-3, IL-6, G-SCF, TPO) promotes hematopoiesis within embryoid aggregates and may result in the appearance of tissue-like structures such as blood islands and early blood vessels. The combination of BMP4 with hematopoietic cytokines yields up to 20% of CD34+CD45+ cells that will give rise to erythroid, macrophage, granulocytic and megakaryocytic colonies. To produce EBs of equal size and standardize differentiation, a certain number of cells can be used to form aggregates by a spin technique (centrifugation) or in a hanging drops method. Hanging drops are single 10-20μl drops with known cell densities that are inoculated into micro-wells. Several studies have shown improvements of this method that would allow it practical application.

Coculture with stromal cells

This two-dimensional differentiation system is based on induction of hematopoiesis upon exposure to extrinsic signals from the feeder cells that underlie the PSCs in coculture. Stromal cells with the capacity to induce and support hematopoiesis can be isolated from a variety of anatomical sites associated with the hematopoietic development in vivo. A number of cell lines were established from mouse bone marrow (OP9, MS5 and S17), yolk sac endothelium (C166), fetal liver (mFLSC, EL08) and other sources. The genetically modified stromal cells, immortalized or expressing specific growth factors and signaling molecules are widely used in hematopoietic coculture. The standard coculture conditions comprise prolonged, up to 4 weeks, incubation of undifferentiated pluripotent stem cells on top of the stromal cells in the presence of fetal bovine serum (FBS) and/or hematopoietic cytokines. Both mouse and human pluripotent cells can be successfully differentiated into CD34+ multi-lineage blood progenitors in a coculture, though the efficiency of hematopoietic differentiation significantly varies between different stromal cell lines and compositions of differentiation media.

Defined feeder-free, serum-free systems

stem_cells

These systems designed to avoid the usage of undefined, animal-origin components such as FBS and stroma cells to achieve highly reproducible and efficient outputs. Thus, PSCs can be plated on matrix protein collagen IV and differentiated into primitive CD34+CD43+ hematopoietic progenitors by exposure to BMP4, bFGF and VEGF. This initial differentiation is more efficient when accompanied with the hypoxic conditions (5% oxygen tension) that resemble the environment of a developing embryo. A further incubation of blood progenitors with the various combinations of cytokines yields maturation of CD71+CD235a+ erythroid cells, CD41a+ CD42b+ megakaryocytes, HLA-DR+CD1a+ dendritic cells, CD14+CD68+ macrophages, CD45+CD117+ mast cells and CD15+CD66+ neutrophils. Despite the great progress achieved in the in vitro modeling of hematopoiesis, blood production from PSCs is still a variable process.  The final goal of intensive research in this area – a consistent production of engraftable cells, capable of reconstitution of all blood lineages in a body, remains a major challenge. Finding critical intrinsic and extrinsic factors that can recreate the unique properties of a hematopoietic stem cell niche in vitro, could advance the generation and expansion of PSCs-derived hematopoietic stem cells in the future.

 

The history of Next Generation Sequencing (NGS).

The history of Next Generation Sequencing (NGS).

by | Mar 7, 2017 | Bioinformatics

Since the deoxyribonucleic acid (DNA) was discovered by Oswald Avery in 1944 and its double helical strand structure composed of four bases was determined by Watson and Crick in 1953, our knowledge about genome structure and function has tremendously increased. This knowledge and its multiple applications, such as molecular cloning, breeding, finding pathogenic genes, comparative and evolution studies, would not exist without sequencing technologies.

First sequencing techniques were developed in 1977 by Frederic Sanger and Walter Gilbert. Sanger sequencing, based on the chain termination method, was adopted as a primary technique in the “first generation” of laboratory and commercial sequencing applications. At that time, DNA sequencing was a labor consuming and required radioactive materials. A decade later, in 1987, Applied Biosystems (AB) introduced the first automatic sequencing machine (AB370), utilizing capillary electrophoresis, which made the sequencing process much faster and accurate. Thus, the capillary sequencing machines and Sanger sequencing technology became the main tools for the completion of Human Genome Project in 2003.

This project and X-PRIZE competition stimulated the development of the next or second-generation of sequencing applications (NGS) that exhibit massively parallel analysis, high throughput, and reduced cost. Massive parallel sequencing utilizes simultaneous reading of a large amount  (1-100 million) of short, 50-400 bases, DNA fragments. Years of evolution yielded three major sequencing systems: i) Roche 454 System- detection of pyrophosphate released during nucleotide incorporation; ii) AB sequencing by Oligo Ligation Detection (SOLiD), iii) Illumina GA/HiSequ System that is based on Solexa’s Genome Analyzer (GA) – sequencing by synthesis (SBS). These highly effective sequencing systems demonstrate their own advantages in terms of read length, accuracy and cost. Briefly, the Illumina HiSeq 2000 features the biggest output and lowest reagent cost, the SOLiD system has the highest accuracy and the Roche 454 system has the longest read length. Other advanced sequencing systems include compact Personal Genome Machine (PGM) and MiSeq, targeted for clinical applications and small laboratories.

While the second generation of sequencing became widely popular in basic research, new improvements in sequencing technologies open era of the third generation sequencers which have two main characteristics: i) PCR is not needed before sequencing which shortens DNA preparation time to several hours; ii) the signal, the fluorescence (PacBio) or the electric current (Nanopore), is captured in real time and get monitored during the enzymatic reaction.

Single-molecular real-time (SMRT) is the third-generation of sequencing method developed by Pacific Bioscience (PacBio, Menlo Park, CA, USA). This method has a shorten sample preparation time, longer average read length (1300pb), which is quite useful for the clinical laboratories and microbiological research and allows predicting epigenetic modifications such as DNA methylation due to differential intensity of the fluorescent signal.

Nanopore sequencing method represents another of state-of-the-art bioengineering model. Nanopore is a tiny biopore, a ion-exchange channel within a trans-membrane protein α-haemolysin (α-HL) isolated from Staphylococcus aureus. The unique property of this protein is its tolerance to the extraordinary voltage up to 100mV with current 100pA.  In nanopore sequencing, the ionic flow is applied continuously while the single-stranded DNA thread moves through the pore and disrupts the current.  Detected by the electrophysiological technique fluctuations in current depend on the size of dNMPs and comprise the readout. The outstanding Oxford Nanopore technology allows sequencing your genome “before dinner”.

Among the broad roles gene expression profiling has played in life sciences, it recently has been utilized to functionally characterize biological systems in basic research and discover biomarkers for disease and treatment management in clinical settings. The NuGEN company offers a fast, simple, robust RNA sample preparation solutions broaden the usable sample types for analysis on all leading microarray and NGS analysis platforms. Whether samples are limited and degraded, or ample and well-preserved, NuGEN’s RNA sample preparation technologies provide solutions of superior sensitivity for rapid analysis of the transcriptome. Whether the focus is RNA-Seq, 3′ or whole transcriptome analysis requiring single-stranded or double-stranded product, there are systems that meet those requirements.