Isolation and therapeutic potential of human haemopoietic stem cells

The haemopoietic stem cell (HSC) has long been regarded as an archetypal, tissue specific, stem cell, capable of completely regenerating haemopoiesis after myeloablation. It has proved relatively easy to harvest HSC, from bone marrow or peripheral blood. In turn, isolation of these cells has allowed therapeutic stem cell transplantation protocols to be developed, that capitalise on their prodigious self renewal and proliferative capabilities. Ex vivo approaches have been described to isolate, genetically manipulateand expand pluripotent stem cell subsets. These techniques have been crucial to the development of gene therapy, and may allow adults to enjoy the potential advantages of cord blood transplantation. Recently, huge conceptual changes have occurred in stem cell biology. In particular, the dogma that, in adults, stem cells are exclusively tissue restricted has been questioned and there is great excitement surrounding the potential plasticity of these cells, with the profound implications that this has, for developing novel cellular therapies. Mesenchymal stem cells, multipotent adult progenitor cells and embryonic stem cells are potential sources of cells for transplantation purposes. These cells may be directed toproduce HSC, in vitro and in the future may be used for therapeutic, or drug development, purposes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Stem cells: their source,potency and use in regenerative therapies with focus on adipose-derived stem cells – a review

Stem cells can be defined as units of biological organization that are responsible for the development and the regeneration of organ and tissue systems. They are able to renew their populations and to differentiate into multiple cell lineages. Therefore, these cells have great potential in advanced tissue engineering and cell therapies. When seeded on synthetic or nature-derived scaffolds in vitro, stem cells can be differentiated towards the desired phenotype by an appropriate composition, by an appropriate architecture, and by appropriate physicochemical and mechanical properties of the scaffolds, particularly if the scaffold properties are combined with a suitable composition of cell culture media, and with suitable mechanical, electrical or magnetic stimulation. For cell therapy, stem cells can be injected directly into damaged tissues and organs in vivo. Since the regenerative effect of stem cells is based mainly on the autocrine production of growth factors, immunomodulators and other bioactive molecules stored in extracellular vesicles, these structures can be isolated and used instead of cells for a novel therapeutic approach called “stem cell-based cell-free therapy”. There are four main sources of stem cells, i.e. embryonic tissues, fetal tissues, adult tissues and differentiated somatic cells after they have been genetically reprogrammed, which are referred to as induced pluripotent stem cells (iPSCs). Although adult stem cells have lower potency than the other three stem cell types, i.e. they are capable of differentiating into only a limited quantity of specific cell types, these cells are able to overcome the ethical and legal issues accompanying the application of embryonic and fetal stem cells and the mutational effects associated with iPSCs. Moreover, adult stem cells can be used in autogenous form. These cells are present in practically all tissues in the organism. However, adipose tissue seems to be the most advantageous tissue from which to isolate them, because of its abundancy, its subcutaneous location, and the need for less invasive techniques. Adipose tissue-derived stem cells (ASCs) are therefore considered highly promising in present-day regenerative medicine.     

Mechanism and methods to induce pluripotency

Pluripotent stem cells are able to self-renew indefinitely and differentiate into all types of cells in the body. They can thus be an inexhaustible source for future cell transplantation therapy to treat degenerative diseases which currently have no cure. However, non-autologous cells will cause immune rejection. Induced pluripotent stem cell (iPSC) technology can convert somatic cells to the pluripotent state, and therefore offers a solution to this problem. Since the first generation of iPSCs, there has been an explosion of relevant research, from which we have learned much about the genetic networks and epigenetic landscape of pluripotency, as well as how to manipulate genes, epigenetics, and microRNAs to obtain iPSCs. In this review, we focus on the mechanism of cellular reprogramming and current methods to induce pluripotency. We also highlight new problems emerging from iPSCs. Better understanding of the fundamental mechanisms underlying pluripotenty and refining the methodology of iPSC generation will have a significant impact on future development of regenerative medicine.     

Induced pluripotent stem (iPS) cells from human fetal stem cells (hFSCs)

Introduction

(1) Human embryonic stem (ES) cells are pluripotent but are difficult to be used for therapy because of immunological, oncological and ethical barriers. (2) Pluripotent cells exist in vivo, i.e., germ cells and epiblast cells but cannot be isolated without sacrificing the developing embryo. (3) Reprogramming to pluripotency is possible from adult cells using ectopic expression of OKSM and other integrative and non-integrative techniques. (4) Hurdles to overcome include i.e stability of the phenotype in relation to epigenetic memory.

Sources of data

We reviewed the literature related to reprogramming, pluripotency and fetal stem cells.

Areas of agreement

(1) Fetal stem cells present some advantageous characteristics compared with their neonatal and postnatal counterparts, with regards to cell size, growth kinetics, and differentiation potential, as well as in vivo tissue repair capacity. (2) Amniotic fluid stem cells are more easily reprogrammed to pluripotency than adult fibroblast. (3) The parental population is heterogeneous and present an intermediate phenotype between ES and adult somatic stem cells, expressing markers of both.

Areas of controversy

(1) It is unclear whether induced pluripotent stem (iPS) derived from amniotic fluid stem cells are fully or partially reprogrammed. (2) Optimal protocols to ensure highest efficiency and phenotype stability remains to be determined. (3) The “level” of reprogramming, fully vs partial, of iPS derived from amniotic fluid stem cells remain to be determined.

Growing points

Banking of fully reprogrammed cells may be important both for (1) autologous and allogenic applications in medicine, and (2) disease modeling.     

A comment on the Hill–Turney exchange: from normative antagonism to interdisciplinary collaboration

Amniotic fluid-derived stem (AFS) cells were the first non-embryonic source of pluripotent stem cells discussed in Italy. AFS cells have been defined “ethical stem cells”, a socio-technical label denoting an array of cellular reprogramming techniques, biological artifacts, and somatic stem cells which make it possible to obtain pluripotent stem cells while avoiding the use of human embryos. The alleged pluripotent status of these cells triggered a debate in which pluripotency was re-positioned as the most promising feature of stem cells. This debate was characterized by discursive articulations in which the boundary between multipotency and pluripotency was blurred and a discourse on a duopoly of pluripotency (pertaining both to embryonic and non-embryonic stem cells) ensued. Drawing on qualitative discourse analysis of articles in scientific journals and newspapers, this paper explores the cultural meaning and political uses of these discursive articulations in the Italian stem cell research regulation debate. This paper also examines a set of aporias emerging from the attempt to incorporate ethical stances into the biological ontology of ethical stem cells, and the problem of constructing normativities in the biotechnology field.     

Current challenges in dedifferentiated fat cells research

Dedifferentiated fat cells show great promises as a novel cell source for stem cell research. It has many advantages when used for cell–based therapeutics including abundance, pluripotency, and safety. However, there are many obstacles researchers need to overcome to make the next big move in DFAT cells research. In this review, we summarize the current main challenges in DFAT cells research including cell culture purity, phenotypic properties, and dedifferentiation mechanisms. The common methods to produce DFAT cells as well as the cell purity issue during DFAT cell production have been introduced. Current approaches to improve DFAT cell purity have been discussed. The phenotypic profile of DFAT cells have been listed and compared with other stem cells. Further studies on elucidating the underlying dedifferentiation mechanisms will dramatically advance DFAT cell research.     

Single cell heterogeneity in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) derived from blastocysts and human induced pluripotent stem cells (hiPSCs) generated from somatic cell reprogramming. Due to their self-renewal ability and pluripotent differentiation potential, hPSCs serve as an excellent experimental platform for human development, disease modeling, drug screening, and cell therapy. Traditionally, hPSCs were considered to form a homogenous population. However, recent advances in single cell technologies revealed a high degree of variability between individual cells within a hPSC population. Different types of heterogeneity can arise by genetic and epigenetic abnormalities associated with long-term in vitro culture and somatic cell reprogramming. These variations initially appear in a rare population of cells. However, some cancer-related variations can confer growth advantages to the affected cells and alter cellular phenotypes, which raises significant concerns in hPSC applications. In contrast, other types of heterogeneity are related to intrinsic features of hPSCs such as asynchronous cell cycle and spatial asymmetry in cell adhesion. A growing body of evidence suggests that hPSCs exploit the intrinsic heterogeneity to produce multiple lineages during differentiation. This idea offers a new concept of pluripotency with single cell heterogeneity as an integral element. Collectively, single cell heterogeneity is Janus-faced in hPSC function and application. Harmful heterogeneity has to be minimized by improving culture conditions and screening methods. However, other heterogeneity that is integral for pluripotency can be utilized to control hPSC proliferation and differentiation.     

Allogenic banking of dental pulp stem cells for innovative therapeutics

Medical research in regenerative medicine and cell-based therapy has brought encouraging perspectives for the use of stem cells in clinical trials. Multiple types of stem cells, from progenitors to pluripotent stem cells, have been investigated. Among these, dental pulp stem cells (DPSCs) are mesenchymal multipotent cells coming from the dental pulp, which is the soft tissue within teeth. They represent an interesting adult stem cell source because they are recovered in large amount in dental pulps with non-invasive techniques compared to other adult stem cell sources. DPSCs can be obtained from discarded teeth, especially wisdom teeth extracted for orthodontic reasons. To shift from promising preclinical results to therapeutic applications to human, DPSCs must be prepared in clinical grade lots and transformed into advanced therapy medicinal products (ATMP). As the production of patient-specific stem cells is costly and time-consuming, allogenic biobanking of clinical grade human leukocyte antigen (HLA)-typed DPSC lines provides efficient innovative therapeutic products. DPSC biobanks represent industrial and therapeutic innovations by using discarded biological tissues (dental pulps) as a source of mesenchymal stem cells to produce and store, in good manufacturing practice (GMP) conditions, DPSC therapeutic batches. In this review, we discuss about the challenges to transfer biological samples from a donor to HLA-typed DPSC therapeutic lots, following regulations, GMP guidelines and ethical principles. We also present some clinical applications, for which there is no efficient therapeutics so far, but that DPSCs-based ATMP could potentially treat.     

Current applications of adipose-derived stem cells and their future perspectives简

Adult stem cells have a great potential to treat various diseases. For these cell-based therapies, adipose-derived stem cells (ADSCs) are one of the most promising stem cell types, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs have taken center stage due to their pluripotency. However, ESCs and iPSCs have limitations in ethical issues and in identification of characteristics, respectively. Unlike ESCs and iPSCs, ADSCs do not have such limitations and are not only easily obtained but also uniquely expandable. ADSCs can differentiate into adipocytes, osteoblasts, chondrocytes, myocytes and neurons under specific differentiation conditions, and these kinds of differentiation potential of ADSCs could be applied in regenerative medicine e.g., skin reconstruction, bone and cartilage formation, etc. In this review, the current status of ADSC isolation, differentiation and their therapeutic applications are discussed.     

Direct monitoring of live human pluripotent stem cells by a highly selective pluripotency sensor

Human pluripotent stem cells (hPSCs) are a useful cell source for regenerative medicine. Despite having a potential of hPSCs for cell-based therapy, there is a need for a selective human pluripotency sensor for monitoring of live hPSCs. Here, we report the discovery of a novel pluripotency sensor (SHI5) from BODIPY-based library by high-throughput cell-based screening and describe the use of SHI5 to identify and isolate human embryonic stem cells and human induced pluripotent stem cells. We demonstrate that SHI5-based assay can be applied to live cells that gain pluripotency in the reprogramming process without any effect on their viability. We also show that SHI5 is internalized through a clathrin-mediated endocytosis pathway. These findings suggest that SHI5 can be an attractive sensor for pluripotency cells during reprogramming. Taken together, SHI5-based screening for hPSCs opens probably unlimited possibilities of detection probe for hPSC therapy via assures their safety issue.     

小鼠胚胎干细胞建系技术研究进展

目前,对小鼠胚胎干细胞的研究较为深入,并已成为研究细胞分化及信号转导、新基因发现及功能鉴定、器官发生、人类疾病和药物开发等的有效手段。胚胎干细胞建系是一项基础性工作。虽然技术日趋成熟,有些品系小鼠的胚胎干细胞建系已是常规技术,但不同品系小鼠胚胎干细胞的建系效率仍有很大差异,建系途径和方法各有特点,一个品系胚胎干细胞的建系方法不一定都适用于其他品系。本文从小鼠胚胎干细胞建系的途径、分离操作技术、培养体系等方面进行综述,并就与之相关的有些问题提出思考和对策。     

The prospect of pluripotent stem cell-based therapy

Human embryonic stem cells (hESC) are able to maintain pluripotency in culture, to proliferate indefinitely and to differentiate into all somatic cell types. Due to these unique properties, hESC may become an exceptional source of tissues for transplantation and have a great potential for the therapy of incurable diseases. Here, we review new developments in the area of embryonic stem cells and discuss major challenges — standardization of protocols for cell derivation and cultivation, identification of specific molecular markers, development of new approaches for directed differentiation, etc. — which remain to be settled, prior to safe and successful clinical application of stem cells. We appraise several potential approaches in hESC-based therapy including derivation of autologous cells via therapeutic cloning (1), generation of immune tolerance to allogenic donor cells via hematopoetic chimerism (2), and development of the banks of hESC lines compatible with the main antigens and exhibiting equivalent pluripotency (3). In addition, we discuss briefly induced pluripotent cells, which are derived via genetic modification of autologous somatic cells and are analogous to ESC. Our analysis demonstrates that uncontrollable differentiation in vivo and teratogenic potential of hESC are critical limitations of their application in clinical practice. Therefore, the major approach in hESC therapy is derivation of a specific differentiated progeny, which has lower proliferative potential and immune privilege, yet poses fewer risks for organism. The review demonstrates that cell therapy is far more complex and resource-consuming process as compared with drug-based medicine and consequently pluripotent stem cell biology and technology still requires further investigation and development before these cells can be used in clinical practice.     

Prion potency in stem cells biology

Prion protein (PrP) can be considered a pivotal molecule because it interacts with several partners to perform a diverse range of critical biological functions that might differ in embryonic and adult cells. In recent years, there have been major advances in elucidating the putative role of PrP in the basic biology of stem cells in many different systems. Here, we review the evidence indicating that PrP is a key molecule involved in driving different aspects of the potency of embryonic and tissue-specific stem cells in self-perpetuation and differentiation in many cell types. It has been shown that PrP is involved in stem cell self-renewal, controlling pluripotency gene expression, proliferation, and neural and cardiomyocyte differentiation. PrP also has essential roles in distinct processes that regulate tissue-specific stem cell biology in nervous and hematopoietic systems and during muscle regeneration. Results from our own investigations have shown that PrP is able to modulate self-renewal and proliferation in neural stem cells, processes that are enhanced by PrP interactions with stress inducible protein 1 (STI1). Thus, the available data reveal the influence of PrP in acting upon the maintenance of pluripotent status or the differentiation of stem cells from the early embryogenesis through adulthood.     

Medaka fish stem cells and their applications

Stem cells are present in developing embryos and adult tissues of multicellular organisms. Owing to their unique features, stem cells provide excellent opportunities for experimental analyses of basic developmental processes such as pluripotency control and cell fate decision and for regenerative medicine by stem cell-based therapy. Stem cell cultures have been best studied in 3 vertebrate organisms. These are the mouse, human and a small laboratory fish called medaka. Specifically, medaka has given rise to...     

Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells

BackgroundCell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells.MethodsFused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells.ResultsColonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells.ConclusionsOur results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species.     

The therapeutic potential of stem cells

In recent years, there has been an explosion of interest in stem cells, not just within the scientific and medical communities but also among politicians, religious groups and ethicists. Here, we summarize the different types of stem cells that have been described: their origins in embryonic and adult tissues and their differentiation potential in vivo and in culture. We review some current clinical applications of stem cells, highlighting the problems encountered when going from proof-of-principle in the laboratory to widespread clinical practice. While some of the key genetic and epigenetic factors that determine stem cell properties have been identified, there is still much to be learned about how these factors interact. There is a growing realization of the importance of environmental factors in regulating stem cell behaviour and this is being explored by imaging stem cells in vivo and recreating artificial niches in vitro. New therapies, based on stem cell transplantation or endogenous stem cells, are emerging areas, as is drug discovery based on patient-specific pluripotent cells and cancer stem cells. What makes stem cell research so exciting is its tremendous potential to benefit human health and the opportunities for interdisciplinary research that it presents.     

Human adult pluripotency: Facts and questions

Cellular reprogramming and induced pluripotent stem cell(IPSC) technology demonstrated the plasticity of adult cell fate, opening a new era of cellular modelling and introducing a versatile therapeutic tool for regenerative medicine.While IPSCs are already involved in clinical trials for various regenerative purposes, critical questions concerning their medium-and long-term genetic and epigenetic stability still need to be answered. Pluripotent stem cells have been described in the last decades in various mammalian and human tissues(such as bone marrow, blood and adipose tissue). We briefly describe the characteristics of human-derived adult stem cells displaying in vitro and/or in vivo pluripotency while highlighting that the common denominators of their isolation or occurrence within tissue are represented by extreme cellular stress. Spontaneous cellular reprogramming as a survival mechanism favoured by senescence and cellular scarcity could represent an adaptative mechanism. Reprogrammed cells could initiate tissue regeneration or tumour formation dependent on the microenvironment characteristics. Systems biology approaches and lineage tracing within living tissues can be used to clarify the origin of adult pluripotent stem cells and their significance for regeneration and disease.