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How are Stem Cells Produced for Stem Cell Based Therapies?

How are Stem Cells Produced for Stem Cell Based Therapies?


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I was doing an initial search on google but couldn't come up with anything I'm looking for. I know were stem cells come from and a bit about their levels of differentiation. For example, embryonic stem still becoming mesenchymal stem cells and what not. I also understand that epithelial tissue cells can be converted to a pluripotent stem cell state but at very low efficiency. So, all that aside how are stem cells obtained in the volumes necessary for stem cell based therapies without causing them to start differentiating during production?


I didn't get your question exactly. If you wanna ask how to maintain the potential of stem cells, yes, there are several compound that could keep stem cells, such as Leukemia Inhibitory Factor (LIF). As far I know, the using of pluripotent stem cell, such as ES, iPS, still has the problem of formatting teratoma in vivo studying. It means totally differentiating pluripotent stem cell still need to be work out.


Stem Cells and the Future of Regenerative Medicine (2002)

This report addresses key questions about the biology and therapeutic potential of human stem cells, undifferentiated cells that can give rise to specialized tissues and organs. Medical and scientific interest in stem cells is based on a desire to find a source of new, healthy tissue to treat diseased or injured human organs. It is known that some organs, such as the skin and the liver, are adept at regenerating themselves when damaged, but it is not yet understood why and how some tissues have this capability and others do not. Recent research has indicated that stem cells are a key to these regenerative properties.

There are confirmed sources of stem cells in adult tissues, such as bone marrow, that maintain the ability to differentiate into the diverse cell types of that tissue throughout the life of an organism. However, cells that maintain the ability to divide and differentiate into more specialized cells of different tissue types are rare in the adult. In contrast, the seemingly unlimited potential of the undifferentiated cells of the early embryo has made embryonic stem cells the focus of great scientific interest. Since 1998, when James Thomson of the University of Wisconsin-Madison developed the first human embryonic stem cell (ESC) cultures, increasing attention has been paid to scientific reports hinting at the therapeutic potential of stem cells for treating various degenerative diseases and injuries (Thomson et al., 1998). What is now known as regenerative medicine seeks to understand how and why stem

TABLE 1. Potential US Patient Populations for Stem Cell-Based Therapies

The conditions listed below occur in many forms and thus not every person with these diseases could potentially benefit from stem cell-based therapies. Nonetheless, the widespread incidence of these conditions suggests that stem cell research could help millions of Americans

Source: Derived from Perry (2000).

cells, whether derived from human embryos or adult tissues, are able to develop into specialized tissues, and seeks to harness this potential for tissue-replacement therapies that will restore lost function in damaged organs.

The list of diseases and injuries cited as potential targets of stem cell therapy reveals, in large measure, why stem cells offer so much hope for revolutionary advances in medicine (Table 1). Many of them&mdashsuch as Parkinson&rsquos disease, diabetes, heart disease, Alzheimer&rsquos disease, and spinal cord injury&mdashhave few or no treatment options, so millions of Americans are currently looking for cures.

The hope of using stem cells to produce regenerative therapies poses fundamental questions: Do human ESCs hold all the clinical promise attributed to them? Is realization of that promise imminent? Do stem cells from all sources have the same abilities? What is their potential for regenerative medicine?

THE CHARGE TO THE COMMITTEE

Members of the National Research Council&rsquos Board on Life Sciences and members of the Institute of Medicine&rsquos Board on Neuroscience and Behavioral Health independently decided in December 2000 that they should sponsor a workshop on the scientific and medical value of stem cell research. The Committee on the Biological and Biomedical Applications of Stem Cell Research was appointed to organize the workshop and to produce a report on the biology and biomedical applications of stem cells in regenerative medicine. (Appendix A provides biographical sketches of the committee members.)

The charge to the committee was as follows:

An appointed committee will organize a workshop on the biology and biomedical applications of stem cells. The workshop will examine several aspects of stem cell research, including: the biological properties of stem cells in general, the current state of knowledge about the molecular and cellular controls that govern transdifferentiation in cells originating from different types of tissues, the use of stem cells to generate neurons, heart, kidney, blood, liver and other tissues, and the prospective clinical uses of these tissues. The workshop will consider the biological differences of cells obtained from different sources, for example, embryos, fetal tissues, or adult tissues, and discuss concerns about the use of various sources of stem cells. The committee will produce a report that summarizes the workshop and the scientific and public policy concerns that present both opportunities and barriers to progress in this field.

The committee&rsquos workshop took place on June 22, 2001, at the National Academy of Sciences in Washington, D.C. Appendix B contains the meeting agenda and biographies of the presenters. Audio files of the speakers&rsquo presentations will be available at the workshop Web site: www.nationalacademies.org/stemcells until December 31, 2002.

It is important to explain the limits of the committee&rsquos charge and work. Although data and opinions in the scientific and other scholarly

literature were examined, the project did not attempt an exhaustive review of the scientific literature in this field. It should be noted that shortly after the workshop, the National Institutes of Health released a major report on the &ldquoScientific Progress and Future Research Directions&rdquo of stem cells, and this document has provided valuable information for the committee&rsquos report (NIH, 2001).

The committee organized the workshop to address key issues in the status of stem cell research by gathering information from scientific leaders in the field. In addition, the workshop provided an opportunity for the committee to hear from both those who support embryonic stem cell research and those who oppose it on ethical grounds. The committee did not attempt to resolve the ethical dilemmas and limits its comments to scientific points intended to clarify or inform the ethical discussion. This report synthesizes the workshop presentations and puts forward the committee&rsquos conclusions drawn from that meeting. In particular, the report addresses the following questions:

What characteristics of stem cells make them desirable for regenerative medicine?

Which biological features of stem cells are well established? Which are uncertain?

What implications do the biological features of different stem cells have for the development of therapeutic applications?

What opportunities and barriers does stem cell research face, and how are they relevant to medical therapies?

The committee placed off limits the issue of reproductive cloning, which is sometimes linked to stem cell research because in both cases the somatic cell nuclear transfer (SCNT) technique can be used to create embryos (see Box). The interest in this technique for stem cell research is related to the possibility of producing stem cells for regenerative therapy that are genetically matched to the person needing a tissue transplant. The immune system is poised to reject tissue transplants

Comparison of Stem Cell Production with Reproductive Cloning

The goal of stem cell research using the somatic cell nuclear transfer (SCNT) technique must be sharply contrasted with the goal of reproductive cloning, which, using a similar technique, aims to develop an embryo that is genetically identical with the donor of its genes and then implant that embryo in a woman&rsquos uterus and allow it to mature to birth. Cloning for reproductive purposes will be the subject of a separate report now being developed by the National Academies&rsquo Committee on the Scientific and Medical Aspects of Human Cloning. In the table below, the cellular materials and techniques of stem cell research are compared to that of reproductive cloning.

Adult and Fetal Stem Cells

Embryonic Stem Cells Produced with the SCNT Technique

Reproductive Cloning: Embryos Produced with the SCNT Technique

To obtain undifferentiated stem cells for research and therapy

To obtain undifferentiated stem cells for research and therapy

To obtain undifferentiated stem cells that are genetically matched to recipient for research and therapy

To produce embryo for implantation, leading to birth of a child

Isolated stem cells from adult or fetal tissue

Cells from an embryo at blastocyst stage produced by fertilization

Cells from a blastocyst produced by development of an enucleated egg supplied with nucleus from patient&rsquos somatic cell (SCNT technique)

Enucleated egg supplied with nucleus from donor&rsquos somatic cell (SCNT technique)

Cells produced in culture to replenish diseased or injured tissue

Cells produced in culture to replenish diseased or injured tissue

Cells produced in culture to replenish diseased or injured tissue

Embryo derived from development of egg, implanted and allowed to develop to birth

from genetically non-identical people, and immunological rejection poses serious clinical risks that can be life-threatening. Overcoming the threat of immunological rejection is thus one of the major scientific challenges to stem cell transplantation and, indeed, for transplantations of any sort. The SCNT technique offers the possibility of deriving stem cells for transplantation from the recipient&rsquos own cells. Such cells would produce only the patient&rsquos own proteins and would not cause an immunological reaction when transplanted into that patient.

The committee is respectfully mindful of the wide array of social, political, legal, ethical, and economic issues that must be considered in policy-making in a democracy. And it is impressed by the commitment of all parties in this debate to life and health, regardless of the different conclusions they draw. The committee hopes that, by addressing questions about the scientific potential of stem cell and how that potential can be best realized, it can contribute usefully to the debate and to the enhancement of treatments for disabling human diseases and injuries.

WHAT ARE STEM CELLS? BASIC DEFINITIONS

Stem cells are unspecialized cells that can self-renew indefinitely and that can also differentiate into more mature cells with specialized functions. In humans, stem cells have been identified in the inner cell mass of the early embryo in some tissues of the fetus, the umbilical cord and placenta and in several adult organs. In some adult organs, stem cells can give rise to more than one specialized cell type within that organ (for example, neural stem cells give rise to three cell types found in the brainneurons, glial cells, and astrocytes). Stem cells that are able to differentiate into cell types beyond those of the tissues in which they normally reside are said to exhibit plasticity. When a stem cell is found to give rise to multiple tissue types associated with different organs, the stem cell is referred to as multipotent. 1

The word &ldquopluripotent&rdquo is sometimes used to describe stem cells that can differentiate into a very wide range of tissue types. In this report the term multipotent encompasses this type of stem cell.

Embryonic stem cells (ESCs) are derived from an early-stage embryo. Fertilization of an ovum by a sperm results in a zygote, the earliest embryonic stage (Figure 1). The zygote begins to divide about 30 hours after fertilization and by the third-to-fourth day, the embryo is a compact ball of 12 or more cells known as the morula. Five-to-six days after fertilization, and after several more cycles of cell division, the morula cells begin to specialize, forming a hollow sphere of cells, called a blastocyst, which is about 150 microns in diameter (one-seventh of a millimeter). The outer layer of the blasotocyst is called the trophoblast, and the cluster of cells inside the sphere is called the inner cell mass. At this stage, there are about 70 trophoblast cells and about 30 cells in the inner cell mass. The cells of the inner cell mass are multipotent stem cells that give rise to all cell types of the major tissue layers (ectoderm, mesoderm, and endoderm) of the embryo. In the past 3 years, it has become possible to remove these stem cells from the blastocyst and maintain them in an undifferentiated state in cell culture lines in the laboratory (NIH, 2001) (Figure 2). To be useful for producing medical therapies, cultured ESCs will need to be differentiated into appropriate tissues for transplantation into patients. Researchers are just beginning to learn how to achieve this differentiation.

Fetal stem cells are primitive cell types in the fetus that eventually develop into the various organs of the body, but research with fetal tissue so far has been limited to only a few cell types: neural stem cells, including neural crest cells hematopoietic stem cells and pancreatic islet progenitors. Neural stem cells, which are numerous in the fetal brain, can be isolated and grown in an undifferentiated form in culture, and they have been shown to differentiate into the three main types of brain cells (Brustle et al., 1998 Villa et al., 2000). These cells have been used in rodent models of Parkinson&rsquos disease (Sawamoto et al., 2001 Studer et al., 1998). Neural crest cells arise from the neural tube and migrate from it throughout the developing fetus. They are able to develop into multiple cell types, including the nerves that innervate the heart and the gut, non-neural cells of hormone-secreting glands, pig-


Stem cell-based therapies threatened by the accumulation of p53 mutations

Human embryonic stem (ES) cells can self-renew indefinitely and give rise to virtually all cell types in the body. This makes them a valuable source of cells for regenerative therapies. ES cell-derived differentiated cells are being evaluated in clinical trials for their safety in therapeutic interventions for several diseases. McCarroll, Eggan and colleagues now report that human ES cells accumulate mutations in TP53 — the gene that encodes the tumour suppressor p53 — which is mutated in ∼ 50% of cancers.

Previous studies have shown that cultured human pluripotent stem cells (PSCs) can acquire aneuploidy and large copy number variations, and that these mutations can confer a growth advantage to cells. The authors set out to identify other mutations that could be acquired in culture, by sequencing all exons in the genome (exomes) of 140 independent cultured ES cell lines (114 lines maintained by the US National Institutes of Health and 26 lines prepared under good manufacturing practice conditions for potential clinical use). They then used computational analyses to identify mutations that were present only in a subset of cells in each ES cell line, thus excluding inherited polymorphisms. With this approach, they identified 263 of such candidate mosaic variants, 28 of which were predicted to disrupt gene function.

The authors went on to characterize these 28 mutations and, strikingly, found that six of them were in TP53, which was also the only gene that was mutated more than once. The six mutations were identified in five unrelated cell lines. The six missense mutations (all involving a cytosine residue of a CpG dinucleotide) mapped to four residues in the DNA-binding domain of p53. Mutations at these positions have been shown to be dominant negative, by preventing wild-type p53 from binding to the promoters of its target genes, and are associated with a high risk of developing cancer.

The authors showed that all six mutations were acquired during cell culture, and estimated that they were present in a substantial fraction (14–80%) of cells in the affected cell lines. Analysis of cells from early culture passages confirmed that the TP53 mutations conferred a strong selective advantage, with the proportion of mutant alleles increasing ∼ 1.9-fold per passage. This finding is consistent with previous reports that p53 loss promotes cell survival, proliferation and the reprogramming of somatic cells to pluripotency.

Lastly, an analysis of publically available RNA sequencing data from 117 human PSC lines (that are capable of differentiating into different cell types) revealed another eight missense mutations in TP53 that are distinct from the six identified in the present study, but which are also in the p53 DNA-binding domain. Some of these published studies used the same source cell line, indicating that the mutations also arose during cell culture.

“This study . highlights the need for careful genetic analyses of stem cells and their differentiated derivatives before clinical use”

This study indicates that cultured human PSCs have a high propensity to accumulate cancer-related mutations in TP53, with implications for their use in disease modelling and cell replacement therapies. It demonstrates the need to develop new culture conditions that could reduce the selective pressure for TP53 mutations to occur and, crucially, it highlights the need for careful genetic analyses of stem cells and their differentiated derivatives before clinical use. Importantly, the study also indicates that sequencing can be used to detect potentially harmful mutations and thus increase the safety of cell replacement therapies.


2. Current clinical applications of stem cells

In all the publicity that surrounds embryonic and iPS cells, people tend to forget that stem cell-based therapies are already in clinical use and have been for decades. It is instructive to think about these treatments, because they provide important caveats about the journey from proof-of-principle in the laboratory to real patient benefit in the clinic. These caveats include efficacy, patient safety, government legislation and the costs and potential profits involved in patient treatment.

Haemopoietic stem cell transplantation is the oldest stem cell therapy and is the treatment that is most widely available (Perry & Linch 1996 Austin et al. 2008). The stem cells come from bone marrow, peripheral blood or cord blood. For some applications, the patient's own cells are engrafted. However, allogeneic stem cell transplantation is now a common procedure for the treatment of bone marrow failure and haematological malignancies, such as leukaemia. Donor stem cells are used to reconstitute immune function in such patients following radiation and/or chemotherapy. In the UK, the regulatory framework put in place for bone marrow transplantation has now an extended remit, covering the use of other tissues and organs (Austin et al. 2008).

Advances in immunology research greatly increased the utility of bone marrow transplantation, allowing allograft donors to be screened for the best match in order to prevent rejection and graft-versus-host disease (Perry & Linch 1996). It is worth remembering that organ transplantation programmes have also depended on an understanding of immune rejection, and drugs are available to provide effective long-term immunosuppression for recipients of donor organs. Thus, while it is obviously desirable for new stem cell treatments to involve the patient's own cells, it is certainly not essential.

Two major advantages of haemopoietic stem cell therapy are that there is no need to expand the cells in culture or to reconstitute a multicellular tissue architecture prior to transplantation. These hurdles have been overcome to generate cultured epidermis to provide autologous grafts for patients with full-thickness wounds, such as third-degree burns. Proof-of-principle was established in the mid-1970s, with clinical and commercial applications following rapidly (Green 2008). Using a similar approach, limbal stem cells have been used successfully to restore vision in patients suffering from chemical destruction of the cornea (De Luca et al. 2006).

Ex vivo expansion of human epidermal and corneal stem cells frequently involves culture on a feeder layer of mouse fibroblastic cells in medium containing bovine serum. While it would obviously be preferable to avoid animal products, there has been no evidence over the past 30 years that exposure to them has had adverse effects on patients receiving the grafts. The ongoing challenges posed by epithelial stem cell treatments include improved functionality of the graft (e.g. through generation of epidermal hair follicles) and improved surfaces on which to culture the cells and apply them to the patients. The need to optimize stem cell delivery is leading to close interactions between the stem cell community and bioengineers. In a recent example, a patient's trachea was repaired by transplanting a new tissue constructed in culture from donor decellularized trachea seeded with the patient's own bone marrow cells that had been differentiated into cartilage cells (Macchiarini et al. 2008).

Whereas haemopoietic stem cell therapies are widely available, treatments involving cultured epidermis and cornea are not. In countries where cultured epithelial grafts are available, the number of potential patients is relatively small and the treatment costly. Commercial organizations that sell cultured epidermis for grafting have found that it is not particularly profitable, while in countries with publicly funded healthcare the need to set up a dedicated laboratory to generate the grafts tends to make the financial cost�nefit ratio too high (Green 2008).

Clinical studies over the last 10 years suggest that stem cell transplantation also has potential as a therapy for neurodegenerative diseases. Clinical trials have involved grafting brain tissue from aborted foetuses into patients with Parkinson's disease and Huntington's disease (Dunnett et al. 2001 Wright & Barker 2007). While some successes have been noted, the outcomes have not been uniform and further clinical trials will involve more refined patient selection, in an attempt to predict who will benefit and who will not. Obviously, aside from the opposition in many quarters to using foetal material, there are practical challenges associated with availability and uniformity of the grafted cells and so therapies with pure populations of stem cells are an important, and achievable (Conti et al. 2005 Lowell et al. 2006), goal.

No consideration of currently available stem cell therapies is complete without reference to gene therapy. Here, there have been some major achievements, including the successful treatment of children with X-linked severe combined immunodeficiency. However, the entire gene therapy field stalled when several of the children developed leukaemia as a result of integration of the therapeutic retroviral vector close to the LMO2 oncogene locus (Gaspar & Thrasher 2005 Pike-Overzet et al. 2007). Clinical trials have since restarted, and in an interesting example of combined gene/stem cell therapy, a patient with an epidermal blistering disorder received an autologous graft of cultured epidermis in which the defective gene had been corrected ex vivo (Mavilio et al. 2006).

These are just some examples of treatments involving stem cells that are already in the clinic. They show how the field of stem cell transplantation is interlinked with the fields of gene therapy and bioengineering, and how it has benefited from progress in other fields, such as immunology. Stem cells undoubtedly offer tremendous potential to treat many human diseases and to repair tissue damage resulting from injury or ageing. The danger, of course, lies in the potentially lethal cocktail of desperate patients, enthusiastic scientists, ambitious clinicians and commercial pressures (Lau et al. 2008). Internationally agreed, and enforced, regulations are essential in order to protect patients from the dangers of stem cell tourism, whereby treatments that have not been approved in one country are freely available in another (Hyun et al. 2008).


Special Issue Editor

The issue is intended to address the background of modern technology of stem cell identification in respect of optimal candidates used to treat respective diseases. This will include in vitro propagation and identification of stem cells derived from tissue reservoirs, the creation of personally tailored cells out of somatic cells through induced pluripotential state (iPS), and re-differentiation into stem cell precursor-progenitor pool, genetic modifications of stem cells to increase desired capacities as. pro-angiogenic, pro-regenerative, anti-inflammatory, anti-fibrotic genes. This would include all technicalities of genes introduction (transient versus stable gene overexpression), optimization of promoters, vectors, and imaging systems (molecular probes singular/double). Personally tailored own stem cells through iPS technology would include genetic overexpression, epigenetic strategy, mRNA (including small regulatory molecules), proteins, and small chemical molecules (methylation vs demethylation). Cell re-differentiation would include cocktails of growth factors, media components, artificial intelligence automatic systems, accelerated in vitro cell maturation (specifically in case of muscular components including skeletal muscles and heart as well as cells of central nervous system). Stem cell delivery is connected with stem cell monitoring of migratory routes and transitions including novel instrumentation of live in situ tracking systems, endoscopy, ultrasound, isotopic and non-isotopic ways of detection. Stem cell retention in target organs would require chaperones for the other accompanying stem/progenitor cells, gradient creation when using interplay of receptors with chemokine attraction molecules, adjuvating medicines provided by nanotechnological means as nanocapsules with self-degrading properties and resistant to a pro-inflammatory milieu while demonstrating stimulating properties to stem cells action. Moreover, papers that outline materials, nano-materials and scaffolds in combination with both cell retention issues as well as adaptation to microenvironment and organ specificity are welcome. We seek papers on immunomodulatory properties towards stem cell, acceptance in immunoprivileged sites as a part of protocols optimization depending on demand of target organ. Finally, we are looking for papers on organoids from the future perspective of stem cell technology and 3-D organ architecture to finish a futuristic view of organ replacement.

Prof. Dr. Maciej Kurpisz
Guest Editor

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HESC derivation and media

hESCs can be derived using a variety of methods, from classic culturing to laser-assisted methodologies or microsurgery [11]. hESC differentiation must be specified to avoid teratoma formation (see Fig. 3).

Spontaneous differentiation of hESCs causes the formation of a heterogeneous cell population. There is a different result, however, when commitment signals (in forms of soluble factors and culture conditions) are applied and enable the selection of progenitor cells

hESCs spontaneously differentiate into embryonic bodies (EBs) [12]. EBs can be studied instead of embryos or animals to predict their effects on early human development. There are many different methods for acquiring EBs, such as bioreactor culture [13], hanging drop culture [12], or microwell technology [14, 15]. These methods allow specific precursors to form in vitro [16].

The essential part of these culturing procedures is a separation of inner cell mass to culture future hESCs (Fig. 4) [17]. Rosowski et al. [18] emphasizes that particular attention must be taken in controlling spontaneous differentiation. When the colony reaches the appropriate size, cells must be separated. The occurrence of pluripotent cells lasts for 1–2 days. Because the classical utilization of hESCs caused ethical concerns about gastrulas used during procedures, Chung et al. [19] found out that it is also possible to obtain hESCs from four cell embryos, leaving a higher probability of embryo survival. Additionally, Zhang et al. [20] used only in vitro fertilization growth-arrested cells.

Culturing of pluripotent stem cells in vitro. Three days after fertilization, totipotent cells are formed. Blastocysts with ICM are formed on the sixth day after fertilization. Pluripotent stem cells from ICM can then be successfully transmitted on a dish

Cell passaging is used to form smaller clusters of cells on a new culture surface [21]. There are four important passaging procedures.

Enzymatic dissociation is a cutting action of enzymes on proteins and adhesion domains that bind the colony. It is a gentler method than the manual passage. It is crucial to not leave hESCs alone after passaging. Solitary cells are more sensitive and can easily undergo cell death collagenase type IV is an example [22, 23].

Manual passage, on the other hand, focuses on using cell scratchers. The selection of certain cells is not necessary. This should be done in the early stages of cell line derivation [24].

Trypsin utilization allows a healthy, automated hESC passage. Good Manufacturing Practice (GMP)-grade recombinant trypsin is widely available in this procedure [24]. However, there is a risk of decreasing the pluripotency and viability of stem cells [25]. Trypsin utilization can be halted with an inhibitor of the protein rho-associated protein kinase (ROCK) [26].

Ethylenediaminetetraacetic acid (EDTA) indirectly suppresses cell-to-cell connections by chelating divalent cations. Their suppression promotes cell dissociation [27].

Stem cells require a mixture of growth factors and nutrients to differentiate and develop. The medium should be changed each day.

Traditional culture methods used for hESCs are mouse embryonic fibroblasts (MEFs) as a feeder layer and bovine serum [28] as a medium. Martin et al. [29] demonstrated that hESCs cultured in the presence of animal products express the non-human sialic acid, N-glycolylneuraminic acid (NeuGc). Feeder layers prevent uncontrolled proliferation with factors such as leukaemia inhibitory factor (LIF) [30].

First feeder layer-free culture can be supplemented with serum replacement, combined with laminin [31]. This causes stable karyotypes of stem cells and pluripotency lasting for over a year.

Initial culturing media can be serum (e.g. foetal calf serum FCS), artificial replacement such as synthetic serum substitute (SSS), knockout serum replacement (KOSR), or StemPro [32]. The simplest culture medium contains only eight essential elements: DMEM/F12 medium, selenium, NaHCO3, l -ascorbic acid, transferrin, insulin, TGFβ1, and FGF2 [33]. It is not yet fully known whether culture systems developed for hESCs can be allowed without adaptation in iPSC cultures.


Embryonic development in a dish

Recent studies aimed at producing specific differentiated cells from ESCs or iPSCs have followed the principle established by Wichterle and colleagues [12] and attempted to recapitulate embryonic development in cell culture. At the core of this approach is the recognition that embryonic development occurs as a series of steps, with cells that have multipotential capacity becoming increasingly differentiated (Figure 1). However, even armed with this recognition, success has been somewhat mixed.

The most common approach for regulating cell differentiation is based on coaxing cells through sequential stages of differentiation. The top schematic is generic and could be applied to any cell type. The lower paradigm is one that could be used to produce pancreatic β-cells and is taken from the work of Chen et al. [43]. DE, definitive endoderm EP, endocrine progenitor PP, pancreatic progenitor.

One instructive example is that of Kattman and colleagues [27], who published a very thorough paper describing a protocol to produce cardiac myocytes from ESCs and iPSCs in which they sequentially added morphogenic factors important in the appearance of cardiac muscle. They stressed a few general conclusions: (a) the first step of any differentiation procedure, the induction of the correct germ layer, must occur efficiently (b) quantitative markers of different stages of development are helpful (c) the timing of activation or inhibition of various morphogenic pathways is critical, especially given that the very same pathway can have a stimulatory or an inhibitory influence at different times and (d) the concentration of the inducing factors must be controlled carefully. In essence, this work confirms that the complex environment of the embryo can be reproduced to at least some degree. However, the authors also pointed out that there is significant variation among different cell lines so that protocols may have to be tailored to each, perhaps because individual lines may make variable amounts of their own inducing factors. This would be a significant hurdle if it were necessary to produce cardiac myocytes from tens or hundreds of patient lines for drug toxicity testing. Thus, finding a way of overriding this variability would be a valuable advance.

Again by adopting an analogous strategy, Studer and colleagues [28] have pursued methods for producing particular types of neurons efficiently. Importantly, they introduced a convenient way of regulating early neural induction by treating human ESCs, grown without standard feeder layers, with inhibitors of both TGF-β and bone morphogenetic protein (BMP) signaling [28]. This group went on to show the utility of this technique in the generation of dopaminergic neurons and motor neurons. Subsequent studies confirmed its utility in the derivation of cell types as diverse as neural crest [29] and floor plate [30].


How are Stem Cells Produced for Stem Cell Based Therapies? - Biology

Regenerative Medicine encompasses many fields of science and medicine.  The image below effectively portrays the scope of Regenerative Medicine as the umbrella, it covers many fields of research and clinical practice. Stem cell research and therapies continue to enhance the field of Regenerative Medicine and what it offers patients and scientists.  Stem cells have and will continue to play a critical role in scientific discoveries through developmental biology and therapeutic applications, however, we should be mindful to not limit our descriptions or thoughts regarding Regenerative Medicine and it’s capabilities to stem cell research alone.  The only constraints placed around it are the ones we set, as those in the field seek to uncover the intricacies of our biological systems.

Typically, when the term ‘Regenerative Medicine’ arises people automatically think about stem cells, particularly, embryonic stem cells.  Being that embryonic stem cell research is currently a highly debated topic in both the scientific and political field, the assumption that Regenerative Medicine Research only involves embryonic stem cell research can be narrowing to the field and does not allow one to understand its full potential.  While all stem cell work is vital to the advancement of Regenerative Medicine research and therapies, we cannot interchange the two terms as equals.  As we learn more about Regenerative Medicine, we must broaden our minds, so as not to limit the vast possibilities that Regenerative Medicine researchers seek to find in the inherent mysteries of our biological systems.  

How are stem cells and Regenerative Medicine linked? 

As discussed in other portions of this site, Regenerative Medicine is a comprehensive term used to describe the current methods and research employed to revive and/or replace dead or damaged tissue.  A portion of Regenerative Medicine research revolves around the use of stem cells, including embryonic, adult, and induced pluripotent stem cells (iPS), however there are many other resources that are utilized in order to carry out the mission of Regenerative Medicine research. These include transplants, biomaterials, scaffolds, machines and electronics, stimulation pathways, drug therapy, and many others.  This is thoroughly discussed on the ‘What is Regenerative Medicine?’ page. 

Stem cells have a very important role in Regenerative Medicine Research and have many potential applications.  First, because of their role in development and their potential to develop into many different cells types, stem cells are vital to the field of developmental biology.  Developmental biologists seek to uncover what genes and pathways are involved in cell differentiation (how cells develop into specific cell types such as liver, skin, or muscle cells) and how these can be manipulated to create new healthy tissues.  Second, stem cells can be applied to drug testing and development.  New drugs that are developed in Pharma could be safely and effectively tested using differentiated stem cells.  As scientists learn more about how stem cells develop to form new tissue they will be able to apply their knowledge in maintaining differentiated cell types that can be used to test particular drugs.  This method is already underway in the cancer therapy world, where cancer cells and grown in the laboratory for the purpose of testing anti-tumor and chemotherapeutic drugs.  Finally, and of most interest to patients and scientists is the role stem cells will play in Cell-Based Therapy.  These therapies will apply the understanding of stem cell development, differentiation, and maintenance to generate new, healthy tissue for diseases needing transplant or replacement of damaged tissue, such as arthritis, Parkinson's disease, type 1 diabetes, and coronary disease.  Cell therapies may one day be able to replace organ donation and eliminate the issues that accompany it such as rejection and tissue insufficiency.   Although there are still many difficulties surrounding the field of stem cell research and therapy, over the coming decades scientists hope to continue to make discoveries that will enable the potentials of cell-based therapy to become a reality. 


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Now available through our exclusive partnership with bit.bio, and its precise opti-ox cellular reprogramming technology, human iPSC-derived cell models become consistent, scalable and reproducible. Generated in large quantities and varied tissue types, bit.bio’s reprogrammed cell types are an excellent tool to support your high-throughput screening campaigns. Combined with our extensive experience in stem cell culturing and differentiation, you now have access to robust assay quality for your HTS and cell-based assays.

Robust HTS with the New Generation of iPSC-Derived Human Cells

Induced pluripotent stem cells (iPSCs) can be used to generate large numbers of cells of varied tissue types, making them an ideal vehicle for HTS and cell-based discovery screening. bit.bio, formerly ElpisBiomed, has applied deep learning algorithms to accelerate the discovery of methods for the reprogramming of every single cell type in the human body. Reprogrammed from patient-derived iPSCs, bit.bio‘s library of validated human cells delivers consistency, purity, scale, and speed to support robust HTS screening.

Our discovery clients now have a distinct advantage. Combining the physiological relevance of bit.bio’s reprogrammed iPSC-derived human cells with Charles River’s extensive experience with stem cell culturing and differentiation, high content imaging, and assay development, you receive reproducible cell populations and robust assay quality for your HTS and cell-based assays.

Case Study: bit.bio Rapid Differentiation into Functional Neurons

Within 12 days, bit.bio’s ioGlutamatergic Neurons convert into consistent, functional glutamatergic neurons. Cells exhibit neurite outgrowth and express numerous key neuronal markers, including Tbr1, MAP2, vGLUT1, synaptophysin and PSD95. ioGlutamatergic Neurons are programmed to rapidly mature upon revival in a 384-well plates without specialty differentiation media or protocols. This is unlike traditional methods which yield inconsistent numbers and purity of neurons, take over 30 days to achieve the same levels of maturity as the programmed ioGlutamatergic Neurons, and often cannot be performed in multi-well plates. Batch to batch reproducibility and homogeneity create a stable human model for excitatory neuronal activity and disease.

In addition to displaying protein markers consistent with neuronal differentiation, ioGlutamatergic Neurons form functional neural networks as measured by MEA after 2-3 weeks of maturation. This allows identification of compounds that show functional alteration of phenotypes relevant to diseased states. Before the bit.bio solution, this process was very difficult to establish at scale.

IoNEURONS/glut are Suitable for HTS Applications

Displaying relevant markers for differentiation, ioGlutamatergic Neurons also differentiate in high density plates. This allows us to perform high-throughput screening in a physiologically-relevant cell type. Using an assay previously developed to identify the presence of huntingtin expressed at physiological levels, we were able to observe reproducible titrations of compounds shown to lower HTT. We were also able to perform functional follow-up assays, again on endogenous protein, to examine whether compounds were acting via toxicity or nonspecific protein degradation.

Robust and Scalable iPSC Cells for HTS

Learn more about the characterization of human iPSC-derived glutamatergic neurons.


About Us

Stem cell technology has opened huge possibilities for cell therapy and regenerative medicine. With professional scientists and years of experience, Creative Biolabs provides high-quality products and services in the field of stem cell therapy development for customers all over the world.

During the last few years, remarkable progress has been made in gene and cell therapy. Positive proof-of- principle results have been obtained for several diseases, such as adrenoleukodystrophy, hemophilia IX, β-thalassemia, malignant glioblastoma, leukemia and other types of cancer.

Thus, it is expected that several new gene therapy products will enter the clinical arena in the not-so-distant future. With the ability to become many different types of cells, stem cells play a key role in the body's healing process and the regenerative medicine.