The potential therapeutic use of stem cells in cartilage repair

As our population demographics change, osteoarthritis and cartilage defects are becoming more prevalent. The discovery of stems cells and their ability for indefinite regeneration has revolutionised the way cartilage problems are viewed. Tissue engineering has been shown to be the ideal way of repairing articular cartilage lesions, i.e. back to native tissue. Cartilage is an ideal tissue engineering target as it is avascular, aneural and alymphatic. The two main types of stem cells being investigated in chondrogenesis are embryological and mesenchymal stem cells. Research into embryological stem cells has been surrounded by controversy because of ethical, religious and social concerns. We discuss the use of embryological and mesenchymal stem cells in cartilage repair and the various factors involved in the differentiation into chondrocytes. We also discuss commonly used mesenchymal stem cell markers and their limitations.     

Autophagy in stem cells

Autophagy is a highly conserved cellular process by which cytoplasmic components are sequestered in autophagosomes and delivered to lysosomes for degradation. As a major intracellular degradation and recycling pathway, autophagy is crucial for maintaining cellular homeostasis as well as remodeling during normal development, and dysfunctions in autophagy have been associated with a variety of pathologies including cancer, inflammatory bowel disease and neurodegenerative disease. Stem cells are unique in their ability to self-renew and differentiate into various cells in the body, which are important in development, tissue renewal and a range of disease processes. Therefore, it is predicted that autophagy would be crucial for the quality control mechanisms and maintenance of cellular homeostasis in various stem cells given their relatively long life in the organisms. In contrast to the extensive body of knowledge available for somatic cells, the role of autophagy in the maintenance and function of stem cells is only beginning to be revealed as a result of recent studies. Here we provide a comprehensive review of the current understanding of the mechanisms and regulation of autophagy in embryonic stem cells, several tissue stem cells (particularly hematopoietic stem cells), as well as a number of cancer stem cells. We discuss how recent studies of different knockout mice models have defined the roles of various autophagy genes and related pathways in the regulation of the maintenance, expansion and differentiation of various stem cells. We also highlight the many unanswered questions that will help to drive further research at the intersection of autophagy and stem cell biology in the near future.     

Emerging roles of neural stem cells in cerebral cortex development and evolution

Expansion and folding of the cerebral cortex are landmark features of mammalian brain evolution, which are recapitulated during embryonic development. Neural stem cells and their derived germinal cells are coordinated during cerebral cortex development to produce the appropriate amounts and types of neurons. This process is further complicated in gyrencephalic species, where newborn neurons must disperse in the tangential axis to expand the cerebral cortex in surface area. Here, we review advances that have been made over the last decade in understanding the nature and diversity of telencephalic neural stem cells and their roles in cortical development, and we discuss recent progress on how newly identified types of cortical progenitor cell populations may have evolved to drive the expansion and folding of the mammalian cerebral cortex.     

Bioreactor design for clinical-grade expansion of stem cells

The many clinical trials currently in progress will likely lead to the widespread use of stem cell-based therapies for an extensive variety of diseases, either in autologous or allogeneic settings. With the current pace of progress, in a few years' time, the field of stem cell-based therapy should be able to respond to the market demand for safe, robust and clinically efficient stem cell-based therapeutics. Due to the limited number of stem cells that can be obtained from a single donor, one of the major challenges on the roadmap for regulatory approval of such medicinal products is the expansion of stem cells using Good Manufacturing Practices (GMP)-compliant culture systems. In fact, manufacturing costs, which include production and quality control procedures, may be the main hurdle for developing cost-effective stem cell therapies. Bioreactors provide a viable alternative to the traditional static culture systems in that bioreactors provide the required scalability, incorporate monitoring and control tools, and possess the operational flexibility to be adapted to the differing requirements imposed by various clinical applications. Bioreactor systems face a number of issues when incorporated into stem cell expansion protocols, both during development at the research level and when bioreactors are used in on-going clinical trials. This review provides an overview of the issues that must be confronted during the development of GMP-compliant bioreactors systems used to support the various clinical applications employing stem cells.     

Mesenchymal stem cells in arthritic diseases

Mesenchymal stem cells (MSCs), the nonhematopoietic progenitor cells found in various adult tissues, are characterized by their ease of isolation and their rapid growth in vitro while maintaining their differentiation potential, allowing for extensive culture expansion to obtain large quantities suitable for therapeutic use. These properties make MSCs an ideal candidate cell type as building blocks for tissue engineering efforts to regenerate replacement tissues and repair damaged structures as encountered in various arthritic conditions. Osteoarthritis (OA) is the most common arthritic condition and, like rheumatoid arthritis (RA), presents an inflammatory environment with immunological involvement and this has been an enduring obstacle that can potentially limit the use of cartilage tissue engineering. Recent advances in our understanding of the functions of MSCs have shown that MSCs also possess potent immunosuppression and anti-inflammation effects. In addition, through secretion of various soluble factors, MSCs can influence the local tissue environment and exert protective effects with an end result of effectively stimulating regeneration in situ. This function of MSCs can be exploited for their therapeutic application in degenerative joint diseases such as RA and OA. This review surveys the advances made in the past decade which have led to our current understanding of stem cell biology as relevant to diseases of the joint. The potential involvement of MSCs in the pathophysiology of degenerative joint diseases will also be discussed. Specifically, we will explore the potential of MSC-based cell therapy of OA and RA by means of functional replacement of damaged cartilage via tissue engineering as well as their anti-inflammatory and immunosuppressive activities.     

Expansion of human hematopoietic stem cells for transplantation: trends and perspectives

Umbilical cord blood transplantation is clinically limited by its low progenitor cell content. Ex vivo expansion has become an alternative to increase the cell dose available for transplants. Expansion has been evaluated in several ways such as static cultures combining growth factors or mimicking the natural microenvironment using co-culture systems. However, static cultures have a small volume capacity and therefore large-scale expansion has been addressed using bioreactors. These and other biotechnological approaches for the expansion of hematopoietic progenitors and their utility to study several aspects of hematopoietic stem cell biology are discussed here.     

Technology for gene transfer into hematopoietic stem cells (Part 2)]

A hematopoietic stem cell is considered to be one of the ideal targets for gene therapy, and there is expectation that gene therapy will be established based on the technology of hematopoietic stem cell transplantation. However, in recent clinical trials of stem cell gene therapy for monogenic diseases, significant clinical improvement has not been reported. One of the main obstacles is the low efficiency of gene transfer into hematopoietic stem cells. Many investigators have been trying to improve the transduction efficiency to the clinically applicable level. Another approach to solve this problem is to develop the method for selective expansion of transduced hematopoietic stem cells in vivo. We are currently developing novel regulatory genes (selective amplifier genes) for stem cell gene therapy.     

Parthenogenesis-derived multipotent stem cells adapted for tissue engineering applications

Embryonic stem cells are envisioned as a viable source of pluripotent cells for use in regenerative medicine applications when donor tissue is not available. However, most current harvest techniques for embryonic stem cells require the destruction of embryos, which has led to significant political and ethical limitations on their usage. Parthenogenesis, the process by which an egg can develop into an embryo in the absence of sperm, may be a potential source of embryonic stem cells that may avoid some of the political and ethical concerns surrounding embryonic stem cells. Here we provide the technical aspects of embryonic stem cell isolation and expansion from the parthenogenetic activation of oocytes. These cells were characterized for their stem-cell properties. In addition, these cells were induced to differentiate to the myogenic, osteogenic, adipogenic, and endothelial lineages, and were able to form muscle-like and bony-like tissue in vivo. Furthermore, parthenogenetic stem cells were able to integrate into injured muscle tissue. Together, these results demonstrate that parthenogenetic stem cells can be successfully isolated and utilized for various tissue engineering applications.     

The journey of developing hematopoietic stem cells

Hematopoietic stem cells (HSCs) develop during embryogenesis in a complex process that involves multiple anatomical sites. Once HSC precursors have been specified from mesoderm, they have to mature into functional HSCs and undergo self-renewing divisions to generate a pool of HSCs. During this process, developing HSCs migrate through various embryonic niches, which provide signals for their establishment and the conservation of their self-renewal ability. These processes have to be recapitulated to generate HSCs from embryonic stem cells. Elucidating the interactions between developing HSCs and their niches should facilitate the generation and expansion of HSCs in vitro to exploit their clinical potential.     

Long-term self-renewal of postnatal muscle-derived stem cells

The ability to undergo self-renewal is a defining characteristic of stem cells. Self-replenishing activity sustains tissue homeostasis and regeneration. In addition, stem cell therapy strategies require a heightened understanding of the basis of the self-renewal process to enable researchers and clinicians to obtain sufficient numbers of undifferentiated stem cells for cell and gene therapy. Here, we used postnatal muscle-derived stem cells to test the basic biological assumption of unlimited stem cell replication. Muscle-derived stem cells (MDSCs) expanded for 300 population doublings (PDs) showed no indication of replicative senescence. MDSCs preserved their phenotype (ScaI+/CD34+/desmin(low)) for 200 PDs and were capable of serial transplantation into the skeletal muscle of mdx mice, which model Duchenne muscular dystrophy. MDSCs expanded to this level exhibited high skeletal muscle regeneration comparable with that exhibited by minimally expanded cells. Expansion beyond 200 PDs resulted in lower muscle regeneration, loss of CD34 expression, loss of myogenic activity, and increased growth on soft agar, suggestive of inevitable cell aging attributable to expansion and possible transformation of the MDSCs. Although these results raise questions as to whether cellular transformations derive from cell culturing or provide evidence of cancer stem cells, they establish the remarkable long-term self-renewal and regeneration capacity of postnatal MDSCs.     

R‐spondin 1 and noggin facilitate expansion of resident stem cells from non‐damaged gallbladders

Pioneering studies within the last few years have allowed the in vitro expansion of tissue‐specific adult stem cells from a variety of endoderm‐derived organs, including the stomach, small intestine, and colon. Expansion of these cells requires activation of the receptor Lgr5 by its ligand R‐spondin 1 and is likely facilitated by the fact that in healthy adults the stem cells in these organs are highly proliferative. In many other adult organs, such as the liver, proliferating cells are normally not abundant in adulthood. However, upon injury, the liver has a strong regenerative potential that is accompanied by the emergence of Lgr5‐positive stem cells; these cells can be isolated and expanded in vitro as organoids. In an effort to isolate stem cells from non‐regenerating mouse livers, we discovered that healthy gallbladders are a rich source of stem/progenitor cells that can be propagated in culture as organoids for more than a year. Growth of these organoids was stimulated by R‐spondin 1 and noggin, whereas in the absence of these growth factors, the organoids differentiated partially toward the hepatocyte fate. When transplanted under the liver capsule, gallbladder‐derived organoids maintained their architecture for 2 weeks. Furthermore, single cells prepared from dissociated organoids and injected into the mesenteric vein populated the liver parenchyma of carbon tetrachloride‐treated mice. Human gallbladders were also a source of organoid‐forming stem cells. Thus, under specific growth conditions, stem cells can be isolated from healthy gallbladders, expanded almost indefinitely in vitro, and induced to differentiate toward the hepatocyte lineage.     

Intestinal stem cells and stem cell-based therapy for intestinal diseases

Currently, many gastrointestinal diseases are a major reason for the increased mortality rate of children and adults every year. Additionally, these patients may cope with the high cost of the parenteral nutrition (PN), which aids in the long-term survival of the patients. Other treatment options include surgical lengthening, which is not sufficient in many cases, and intestinal transplantation. However, intestinal transplantation is still accompanied by many challenges, including immune rejection and donor availability, which may limit the transplant’s success. The development of more safe and promising alternative treatments for intestinal diseases is still ongoing. Stem cell-based therapy (SCT) and tissue engineering (TE) appear to be the next promising choices for the regeneration of the damaged intestine. However, suitable stem cell source is required for the SCT and TE process. Thus, in this review we discuss how intestinal stem cells (ISCs) are a promising cell source for small intestine diseases. We will also discuss the different markers were used to identify ISCs. Moreover, we discuss the dominant Wnt signaling pathway in the ISC niche and its involvement in some intestinal diseases. Additionally, we discuss ISC culture and expansion, which are critical to providing enough cells for SCT and TE. Finally, we conclude and recommend that ISC isolation, culture and expansion should be considered when SCT is a treatment option for intestinal disorders. Therefore, we believe that ISCs should be considered a cell source for SCT for many gastrointestinal diseases and should be highlighted in future clinical applications.     

Engineering stem cell niches in bioreactors

Stem cells, including embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells and amniotic fluid stem cells have the potential to be expanded and differentiated into various cell types in the body. Efficient differentiation of stem cells with the desired tissue-specific function is critical for stem cell-based cell therapy, tissue engineering, drug discovery and disease modeling. Bioreactors provide a great platform to regulate the stem cell microenvironment, known as “niches”, to impact stem cell fate decision. The niche factors include the regulatory factors such as oxygen, extracellular matrix (synthetic and decellularized), paracrine/autocrine signaling and physical forces (i.e., mechanical force, electrical force and flow shear). The use of novel bioreactors with precise control and recapitulation of niche factors through modulating reactor operation parameters can enable efficient stem cell expansion and differentiation. Recently, the development of microfluidic devices and microbioreactors also provides powerful tools to manipulate the stem cell microenvironment by adjusting flow rate and cytokine gradients. In general, bioreactor engineering can be used to better modulate stem cell niches critical for stem cell expansion, differentiation and applications as novel cell-based biomedicines. This paper reviews important factors that can be more precisely controlled in bioreactors and their effects on stem cell engineering.     

Inoculation and growth conditions for high-cell-density expansion of mammalian neural stem cells in suspension bioreactors

Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (NSC) have been determined. We examined suspension culture bioreactors of murine NSC, and concluded that the oxygen level should be kept high (20%), and the osmolarity of the medium should be kept low (below 400 mOsm/kg). The pH of the medium was found to have a large effect on cell proliferation, and the best growth characteristics were obtained within an optimum pH range of 7. 1 to 7.5. The inoculation conditions were also seen to have a large effect not only on the growth characteristics, but also on the number of cells that die in the initial stages of the culture. For large expansion of cells, low inoculum levels (10(4) cells/mL) and single-cell suspensions proved superior, whereas, for fast expansion of cells, higher inoculum levels (10(5) cells/mL) and spheroid inoculum forms were preferred. The inoculum temperature of the medium did not have a large effect on growth characteristics, but the pH greatly influenced cell proliferation. Inoculum pH levels should also be kept between 7.1 and 7.5. If these protocols are followed, high multiplication ratios and viabilities can be obtained in a 5-day batch suspension culture bioreactor run. A large number of cells could then be used in animal models for testing of neural drugs and in research and development toward cures for neurodegenerative disorders such as multiple sclerosis (MS) and Huntington's and Parkinson's disease. The results presented here also point the way toward studies on in vitro expansion of human neural stem cells.     

Human adipose stem cells: current clinical applications

Adipose-derived stem cells are multipotent cells that can easily be extracted from adipose tissue, are capable of expansion in vitro, and have the capacity to differentiate into multiple cell lineages, which have the potential for use in regenerative medicine. However, several issues need to be studied to determine safe human use. For example, there are questions related to isolation and purification of adipose-derived stem cells, their effect on tumor growth, and the enforcement of U.S. Food and Drug Administration regulations. Numerous studies have been published, with the interest in the potential for regenerative medicine continually growing. Several clinical trials using human adipose stem cell therapy are currently being performed around the world, and there has been a rapid evolution and expansion of their number. The purpose of this article was to review the current published basic science evidence and ongoing clinical trials involving the use of adipose-derived stem cells in plastic surgery and in regenerative medicine in general. The results of the studies and clinical trials using adipose-derived stem cells reported in this review seem to be promising not only in plastic surgery but also in a wide variety of other specialties. Nevertheless, those reported showed disparity in the way adipose-derived stem cells were used. Further basic science experimental studies with standardized protocols and larger randomized trials need to be performed to ensure safety and efficacy of adipose-derived stem cells use in accordance with U.S. Food and Drug Administration guidelines.     

Gene delivery to adult neural stem cells

Neural stem cells may present an ideal route for gene therapy as well as offer new possibilities for the replacement of neurons lost to injury or disease. However, it has proved difficult to express ectopic genes in stem cells. We report methods to introduce genes into adult neural stem cells using viral and nonviral vectors in vitro and in vivo. Adenoviral and VSV-G-pseudotyped retroviral vectors are more efficient than plasmid transfection or VSV-G lentiviral transduction in vitro. We further show that adult neural stem cells can be directed to a neuronal fate by ectopic expression of neurogenin 2 in vitro. Plasmids can be delivered in vivo when complexed with linear polyethyleneimine, and gene expression can be targeted specifically to neural stem or progenitor cells by the use of specific promoters. These techniques may be utilized both to study the function of various genes in the differentiation of neural stem cells to specific cell fates and, ultimately, for gene therapy or to generate specific differentiated progeny for cell transplantation.