Induced stem cells

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Induced stem cells (iSC) are stem cells artificially derived from some other (somatic, reproductive, pluripotent etc.) cell types by induced (i.e. initiated, forced) epigenetic reprogramming. In accordance to the developmental potentiality and the degree of cell dedifferentiation caused by induced reprogramming they are distinguished and subdivided as: induced totipotent, induced pluripotent stem cells (iPSc) and, obtained by so-called direct reprogramming or directed forced differentiation, induced progenitor (multipotent or unipotent) stem cells, also called induced somatic stem cells. Currently, there are three ways to reprogram somatic cells into stem cells[1] These are:

  1. Transplantation of nuclei taken from somatic cells into a fertilized egg or oocyt from which the nucleus is removed prior[2] or, in the case of human eggs, where the removal of the nucleus breaks further division of the oocyte, just fertilized egg with the nucleus;[3][4]
  2. Fusion of somatic cells with pluripotent stem cells[5] and
  3. Modification of somatic cells, inducing its transformation into a stem cell, using: the genetic material encoding reprogramming protein factors,[6][7],;[8] recombinant proteins;[9] microRNA,[10][11][12][13][14] and low-molecular biologically active substances,.[15][16][17][18]

Contents

Metaplasia and Transdetermination - natural processes of induction [edit]

The reversible transformation of one differentiated cell type to another type of mature differentiated cells is called metaplasia.[19] This transition from one cell type to another can be a part of the normal maturation process, or caused by some of its inducing stimulus. For example: transformation of cells of the iris to the lens in the process of maturation and transformation of the retinal pigment epithelium cells into the neural retina during regeneration in adult newt eyes. This process allows the body to replace the original cells not suitable to new conditions, into new cells which are more suited to new conditions. In experiments on cells in Drosophila imaginal discs, it was found that there are a limited number of standard discrete states of differentiation and the cells have to choose one of them. The fact that transdetermination (change of the path of differentiation) often take place not in one, but in a group of cells shows that it is not caused by a mutation but is induced.[20][21]

Induced totipotent cells usually can be obtained by reprogramming somatic cells by somatic-cell nuclear transfer (SCNT).

Induced totipotent cells and their use for cloning and generation of genetically modified animals [edit]

Induced totipotent cells usually can be obtained by reprogramming somatic cells by somatic-cell nuclear transfer (SCNT) to the recipient eggs or oocytes.[4][22][23][24] Sometimes may be used the oocytes of other species, such as sheep.[25] New possibilities for creating genetically modified animals opens method of induced androgenetic haploid embryonic stem cells, which can be used instead of sperm. These cells, synchronized in M phase and injected into the oocyte allow to get viable offspring.[26] These developments, together with data on the possibility to obtain unlimited number of oocytes from mitotically active reproductive stem cells[27] offer the possibility of industrial production of transgenic farm animals. Repeated recloning of viable mice through a somatic cell nuclear transfer method that includes a histone deacetylase inhibitor – trichostatin, added to the cell culture medium,[28] show that it may be possible to reclone animals indefinitely without any visible accumulation of reprogramming or genomic errors [29] However, research into technologies to develop sperm and egg cells from stem cells bring up bioethical issues.

Such technologies may also have far-reaching clinical applications for overcoming cytoplasmic defects in human oocytes.[30][31][32] For example, the technology have been developed that could prevent inherited mitochondrial disease being passed on to the next generation. Mitochondria, often described as the powerhouse of the cell, contain genetic material, which is passed from mother to child. Mutations on mitochondrial DNA can cause diabetes, deafness, eye disorders, gastrointestinal disorders, heart disease, dementia and several other neurological diseases. The nucleus from one human egg cell have been transferred to another egg, effectively swapping the cell cytoplasm, which includes the mitochondria (and their DNA), creating a cell that could be regarded as having two mothers. The eggs were then fertilised, and the resulting embryonic stem cells carried the swapped mitochondrial DNA.[33]

iPSc as a result of radical rejuvenation [edit]

See also main article: induced pluripotent stem cells (iPSc)

First iPSc were obtained in the form of transplantable teratocarcinoma induced by the graft taken from mouse embryos.[34] It was shown that teratocarcinoma formed from somatic cells.[35] The fact that normal genetically mosaic mice can be obtained from malignant teratocarcinoma cells confirmed their pluripotency.[36][37][38] It turned out that teratocarcinoma cells are able to maintain a culture of pluripotent embryonic stem cells in an undifferentiated state, by supplying the culture medium with various factors.[39] Thus, as early as in the 1980s, it became clear that the transplantation of pluripotent or embryonic stem cells into the body of adult mammals, usually leads to the formation of teratomas, which can then turn into a malignant tumor teratocarcinoma.[40] If, however, to put the teratocarcinoma cells into the early mammal embryo (at the blastocyst stage), they became incorporated in the cell mass of blastocysts and from such a chimeric (ie composed of cells from different organisms) blastocyst often develops normal chimeric animal.[41][42][43] This indicated that the cause of the teratoma is a dissonance - mutual misunderstanding of "speech" of young donor cells and surrounding adult cells (so-called niche) of the recipient.

In August 2006, Japanese researchers circumvented the need for an oocyte, which is required for SCNT-mediated nuclear reprogramming method. By reprograming mouse embryonic fibroblasts into pluripotent stem cell via the ectopic expression of only four transcription factors, namely Oct4, Sox2, Klf4 and c-Myc, they proved that the overexpression of a surprisingly small number of factors can sometimes push the cell to transition to a new stable state that is associated with changes in the activity of thousands of genes.[6] The properties of IPSC were very similar to embryonic stem cells (ESCs). iPSCs have been shown to support the development of all-iPSC mice using tetraploid (4n) embryo,[44][45] the most stringent assay for developmental potential. However, some genetically normal iPSCs failed to give rise to all-iPSC mice on the score of aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster.[16] An important advantage of iPSC to ESC is that they can be derived from adult cells, rather than from embryos. Therefore, it became possible to obtain iPSC from adults and even elderly patients[8] Reprogramming of somatic cells to iPSC leads to rejuvenation. It was found that reprogramming to iPSC leads to telomere lengthening and shortening after their subsequent differentiation back into fibroblast-like derivatives.[46] Thus, reprogramming of somatic cells to iPSC leads to the restoration of embryonic telomere length,[47] and hence increases the potential number of cell divisions limited by Hayflick limit,.[48][49] Therefore, technology of iPSC should be seen as a radical way to rejuvenation. Unfortunately, because of the dissonance between rejuvenated cells and their surrounding (so-called niche ) of older cells of the recipient, the injection of the patient by his own iPSC, usually leads to an immune response,[50] which can be used for medical purposes,[51] or the formation of tumors such as teratoma.[52] The reason for this probably lies in the fact that some cells differentiated from ESC and iPSC in vivo continue to synthesize the embryonic isoforms of protein.[53]

Hope gives us study which showed that a small molecule called MitoBloCK-6 can force the pluripotent stem cells to die by triggering apoptosis (via cytochrome c release across the mitochondrial outer membrane in human pluripotent stem cells but not in differentiated cells), while shortly after differentiation their daughter cells became resistant to death. When MitoBloCK-6 introduced to differentiated cell lines, the cells remained healthy. The key to the survival of the differentiated cells, when the cells are exposed to MitoBloCK-6, may be due to the changes undergone by pluripotent stem cell mitochondria in the process of cell differentiation. This ability of MitoBloCK-6 to separate the pluripotent and differentiated cell lines could potentially reduce the risk of teratomas and other problems in regenerative medicine treatment strategies.[54] Recently been identified others small molecules (selective cytotoxic inhibitors of hPSCs) that prevented human pluripotent stem cells from forming teratomas in mice following transplantation. The most potent and selective compound of them (PluriSIn #1) inhibits stearoyl-coA desaturase (the key enzyme in oleic acid biosynthesis), which finally results in apoptosis. With the help of this molecule the undifferentiated cells can be selectively removed from culture.[55][56] However, it is unlikely that any kind of preliminary clearance, even the most sophisticated pre-treatment,[57] is able to secure the replanting iPSC or ESC, as after the selective removal of pluripotent cells, they re-emerge quickly by converting differentiated cells back into stem cells, which leads to the formation of tumors,[58][59][60] In this regard, it should be noted that teratoma formation by pluripotent stem cells may be caused by low activity of PTEN enzyme, reported to promote the survival of a small population (0,1-5% of total population) of highly tumorigenic, aggressive, teratoma-initiating embryonic-like carcinoma cells during differentiation. The survival of these teratoma-initiating cells is associated with failed repression of Nanog as well as a propensity for increased glucose and cholesterol metabolism.[61] These teratoma-initiating cells also expressed a lower ratio of p53/p21 when compared to non-tumorigenic cells.[62] In connection with the above safety problems, the use iPSC for cell therapy is still limited.[63] However, they can be used for a variety of other purposes - including the modeling of disease, screening (selective selection) of drugs, toxicity testing of various drugs.[64]

Interestingly, the tissue grown from iPSCs, placed in the "chimeric" embryos in the early stages of mouse development, practically do not cause an immune response (after the embryos have grown into adult mice) and are suitable for autologous transplantation[65][66][67]

Method for producing cells induced to differentiate from iPSCs in the teratoma under in vivo conditions [edit]

The fact that human iPSCs capable of forming teratomas not only in humans but also in some animal body, in particular in mice or pigs, allowed to develop a method for differentiation of iPSCs in vivo. For this purpose, iPSCs with an agent for inducing differentiation into target cells are injected to genetically modified pig or mouse that has suppressed immune system activation on human cells. The formed after a while teratoma is cut out and used for the isolation of the necessary differentiated human cells[68] by means of monoclonal antibody to tissue specific markers on the surface of these cells. This method has been successfully used for the production of functional myeloid, erythroid and lymphoid human cells suitable for transplantation (yet only to mice).[69] Mice engrafted with human iPSC teratoma-derived hematopoietic cells produced human B and T cells capable of functional immune responses. These results offer hope that in vivo generation of patient customized cells is feasible, providing materials that could be useful for transplantation, human antibody generation, and drug screening applications. Using MitoBloCK-6 [54] and / or PluriSIn # 1 the differentiated progenitor cells can be further purified from teratoma forming pluripotent cells. The fact, that the differentiation takes place in a teratoma, offers hope that the resulting cells are sufficiently stable to stimuli able to cause their transition back to the dedifferentiated (pluripotent) state, and therefore safe.

Cell therapy with retina cells obtained from iPSCs [edit]

In the near future will start clinical trials designed to demonstrate the safety of the use of iPSCs for cell therapy of the people with age-related macular degeneration - a disease causing blindness through retina damaging. There are several articles describing methods for producing retinal cells from iPSCs[70] [71] and and how to use them for cell therapy.[72][73] Reports of iPSC-derived retinal pigmented epithelium transplantation showed enhanced visual-guided behaviors of experimental animals for 6 weeks after transplantation.[74]

Differentiation of iPSCs into reproductive cells [edit]

Some lines of iPSCs have the potentiality to differentiate into male germ cells and oocyte-like cells in an appropriate niche (by culturing in retinoic acid and porcine follicular fluid differentiation medium or seminiferous tubule transplantation). Moreover, iPSC transplantation make a contribution to repairing the testis of infertile mice, demonstrating the potentiality of gamete derivation from iPSCs in vivo and in vitro.[75][76][77]

Induced progenitor stem cells [edit]

Methods of direct transdifferentiation [edit]

Due to the fact that the use of iPSC for cell therapy is associated with significant risk of cancer and tumors there is urgent need to develop methods for safer cell lines suitable for use in the clinic. An alternative to the methods of iPSC technique is the so-called "direct reprogramming" - induced by certain factors direct transdifferentiation of cells without passing through the stages of the pluripotent state,.[78][79][80][81][82][83] The basis for this approach laid the study of Taylor and Jones, who showed that 5-azacytidine - reagent causing demethylation of DNA - can cause the formation of myogenic, chondrogenic, and adipogenic clones in the immortal cell line of mouse embryonic fibroblasts[84] and Weintraub et al, who found that the activation of a single gene, later named MyoD1, is sufficient for such reprogramming,[85][86],.[87] Compared with iPSC whose reprogramming need at least two weeks, the formation of induced progenitor cells occurs relatively quickly - sometimes within a few days and the efficiency of reprogramming are usually many times higher. This reprogramming does not always require cell division.[88] But the main thing is that the resulting from such reprogramming multipotent somatic stem cells are more suitable for cell therapy because they do not form teratomas.[83]

Reprogramming by means of a phased process modeling regeneration [edit]

Another way of reprogramming is the simulation of the processes that occur during amphibian limb regeneration. In urodele amphibians, an early step in limb regeneration is skeletal muscle fiber dedifferentiation into a cellulate that proliferates to contribute new limb tissue, whereas, mammalian muscle cannot dedifferentiate after injury. However, sequential small molecule treatment of the muscle fiber with myoseverin, reversine (the aurora B kinase inhibitor) and some other chemicals: BIO (glycogen synthase-3 kinase inhibitor), lysophosphatidic acid (pleiotropic activator of G-protein-coupled receptors), SB203580 (p38 MAP kinase inhibitor), or SQ22536 (adenylyl cyclase inhibitor), causes the formation of new muscle cell types as well as other cell types such as precursors to fat, bone, and nervous system cells,.[89][90]

Transdifferentiation by using an antibody [edit]

The researchers discovered GCSF-mimicking antibody that can activate a growth-stimulating receptor on marrow cells in a way that induces marrow stem cells—which normally develop into white blood cells—to become neural progenitor cells. The new technique,[91] that enables researchers to search large libraries of antibodies and quickly select the ones with a desired biological effect, has been used for such purpose[92][93]

Conditionally reprogrammed cells (CRCs) [edit]

Chapman S. et al.[94] and Liu X. et al.[95] demonstrated a method that can generate adult stem-like cells in vitro without genetic manipulation from human adult keratinocytes and other types of adult epithelial cells, respectively. They show that the combination of irradiated fibroblast feeder cells[96](a review is given in[97] and[98]) and Rho kinase inhibitor, Y-27632,[99][100] conditionally induces an indefinite proliferative state with unknown mechanisms (at least partially due to telomerase induction) in primary mammalian epithelial cells. The induction of CRCs is rapid and results from reprogramming of the entire cell population rather than the selection of a minor subpopulation. CRCs do not express high levels of proteins characteristic of iPSCs or embryonic stem cells (ESCs) (e.g., Sox2, Oct4, Nanog, or Klf4). This induction of CRCs is reversible, and removal of Y-27632 and feeders allows the cells to differentiate normally.[94][95][101] CRC technology can generate 2×106 cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. CRCs retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors.[95][102][103] The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking.[95][102][103] Using conditionally reprogrammed cells technology, researchers were able to identify an effective therapy for a patient with a rare type of lung tumor.[104] In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.

A different approach to obtain conditionally reprogrammed cells is to inhibit CD47 - a membrane protein which is the thrombospondin-1 receptor. It was shown that loss of CD47 permits sustained proliferation of primary murine endothelial cells, increases asymmetric division, and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters. CD47 knockdown acutely increases mRNA levels of c-Myc and other stem cell transcription factors in cells in vitro and in vivo. It is obvious that thrombospondin-1 is a key environmental signal that inhibits stem cell self-renewal via CD47. Thus, CD47 antagonists enable cell self-renewal and reprogramming by overcoming negative regulation of c-Myc and other stem cell transcription factors[105]

Indirect lineage conversion (ILC) [edit]

A reprogramming methodology developed in which somatic cells transition through a plastic intermediate state of partially reprogrammed cells (pre-iPSC), induced by brief exposure to reprogramming factors, followed by differentiation by specially developed chemical environment (artificial niche).[106] It is assumed that this new method could be both more efficient and safer, since it does not seem to produce tumors or other undesirable genetic changes, and results in much greater yield than other methods. However, the safety of these cells all the same questionable - considering that lineage conversion from pre-iPSC relies on the use of iPSC reprogramming conditions, it can not be excluded that a fraction of the cells could acquire pluripotent properties if they do not stop the de-differentation process in vitro or due to further de-differentiation in vivo.

Induced neural stem cells (iNSCs) [edit]

Stroke and many neurodegenerative disorders such as: Parkinson's disease, Alzheimer’s disease, amyotrophic lateral sclerosis need cell replacement therapy. The successful use of converted neural cells (cNs) in transplantations open a new avenue to treat such diseases.[107] Nevertheless, induced neurons (iNs), directly converted from fibroblasts are terminally committed neurons that exhibit very limited proliferative ability and may not provide enough autologous donor cells for transplantation.[108] Additional advantages over iNs towards both basic research and clinical applications provides generation of induced neural stem cells (iNSCs)[81][82][83][109] due to their self-renewal capacity. For example, under specific growth conditions, mouse fibroblasts can be reprogrammed with a single factor, Sox2, to form induced neural stem cells (iNSCs) that self-renew in culture and after transplantation can survive, integrate and do not form tumors in mouse brains.[110] Methods of direct transformation of somatic cells into induced neural stem cells differ in their technique as shown in the review.[111]

Some data provide proof of principle that it is possible, directly in the brain, to convert transplanted human fibroblasts and human astrocytes, which are engineered to express inducible forms of neural reprogramming genes, into neurons, when reprogramming genes are activated after transplantation using a drug in the animals’ drinking water.[112] It was also shown that endogenous mouse astrocytes can be directly converted into neural nuclei (NeuN)-expressing neurons in situ.

Future studies will show which of these approaches proved to be most appropriate for clinic.

Oligodendrocyte precursor cells (OPCs) [edit]

Without myelin to insulate neurons, signals sent down nerve cell axons quickly lose power. Diseases that attack myelin, such as multiple sclerosis, result in nerve signals that are not efficient, because can not propagate to nerve endings, and as a consequence to cognitive, motor and sensory problems. Transplantation of oligodendrocyte precursor cells (OPCs), which can successfully create myelin sheaths around nerve cells, is a promising potential therapeutic strategy for diseases affecting myelin. However, there was no available source of engraftable OPCs. Therefore, direct lineage conversion of mouse and rat fibroblasts into oligodendroglial cells by forced expression of either eight[113] or of the three[114] transcription factors Sox10, Olig2 and Zfp536, laid the groundwork for therapies of a wide array of myelin disorders and spinal cord injury.

Induced cardiomyocytes (iCMs) [edit]

Development of regenerative therapeutic strategies to reverse the progression of advanced heart failure is one of the most urgent clinical needs of this century. Cell-based in vivo therapies may provide a transformative approach to augment vascular and muscle growth and to prevent non-contractile scar formation by delivering transcription factors[78] or microRNAs[13] to the heart.[115] For example: Qian L. et al & Srivastava D. demonstrate that cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of cardiac core transcription factors after coronary ligation.[78] These results implicated therapies that can directly remuscularize the heart without the need for cell transplantation. However, the efficiency of direct cardiac reprogramming in vivo by overexpression of cardiac core transcription factors in cardiac fibroblasts turned out to be very low and phenotype of received cardiomyocyte-like cells does not resemble those of a bona fide mature cardiomyocyte. Furthermore, transplantation of cardiac transcription factors into injured murine hearts resulted in poor cell survival and minimal expression of cardiac genes.[116] So, further technical improvements are needed to make this technology more applicable in situ. Meanwhile, there have been some advances in the methods of obtaining cardiac myocytes in vitro.[117] For example, Carpenter et al. demonstrated efficient cardiac differentiation of human iPS cells that gave rise to progenitors that were retained within the infarcted rat heart, and reduced remodeling of the heart after ischemic damage.[118] Palecek S P et al., have developed a protocol for generating almost pure populations of cardiomyocytes (up to 98% cardiomyocytes) from human pluripotent stem cells simply by modulating canonical Wnt signaling pathway at defined time points in the differentiation process, using readily accessible small molecule compounds.[119] Discovery of the mechanisms controlling the formation of cardiomyocytes led to the development of the drug (ITD-1) which effectively clears the cell surface from TGF-β receptor type II and selectively inhibits the intracellular TGF-β signaling and thus selectively enhances the differentiation of uncommitted mesoderm to cardiomyocytes, but not to vascular smooth muscle and endothelial cells.[120] See also: cell therapy in cardiovascular disease: selected research that has driven recent advances in clinical cardiology

Bioengineering of the cells of blood vessels [edit]

Blood vessels build extensive networks that supply all cells with nutrients and oxygen throughout life. As blood vessels get older, they often become abnormal in structure and function, thereby contributing to numerous age-associated diseases including myocardial infarction, ischemic stroke and atherosclerosis of arteries supplying the heart, brain, and lower extremities. So, an important goal is to stimulate vascular growth for the collateral circulation to prevent the exacerbation of these diseases. A useful cell type for cell-based therapy designed to stimulate coronary collateral growth are Induced Vascular Progenitor Cells (iVPCs), generated by partially reprogramming endothelial cells.[106] The vascular commitment of iVPCs is related to the epigenetic memory of endothelial cells, which engenders them as cellular components of growing blood vessels. That is why, when iVPCs were implanted into myocardium, they engrafted in blood vessels and increased coronary collateral flow better than iPSCs, mesenchymal stem cells, or native endothelial cells.[121] An effective strategy to enhance stem cell function is ex vivo genetic modification. For example, genetic modification with Pim-1 kinase (a downstream effector of Akt, which positively regulates neovasculogenesis) of bone marrow–derived cells[122] or human cardiac progenitor cells, isolated from failing myocardium[123] results in durability of repair, together with the superior improvement of functional parameters of myocardial hemodynamic performance after cellular therapy of the injured myocardium with modified progenitor cells. Stem cells extracted from fat tissue after liposuction may be coaxed into progenitor smooth muscle cells (iPVSMCs) found in arteries and veins.[124] In the future, iVPCs and iPVSMCs may become a necessary source for the creation of blood vessels networks for tissue engineering and reconstruction of organs for transplantation.

Bioengineering of blood stem cells [edit]

Red blood cells (RBC) [edit]

Red blood cells (RBC) transfusion is necessary for many patients with emergency or hematological disorders. However, to date the supply of RBCs remains labile and dependent on voluntary donations. In addition, the transmission of infectious disease via blood transfusion from unspecified donors remains a risk. Establishing a large quantity of safe RBCs would help to address this issue. New technologies for ex vivo erythroid cell generation will hopefully provide alternative transfusion products to meet present and future clinical requirements.[125] In favor of this suggest data that RBCs generated in vitro from mobilized CD34 positive cells have normal survival when transfused into an autologous recipient.[126] Unfortunately, the RBC produced in vitro contained exclusively fetal hemoglobin (HbF) which rescues the functionality of these RBCs. However, in vivo the switch of fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from induced pluripotent stem cells was observed.[127] But then there is another problem: although RBCs do not have nuclei, and therefore can not form a tumor, their immediate precursors the erythroblasts do. The terminal maturation of erythroblasts into functional RBCs requires a complex remodeling process which ends with extrusion of the nucleus and the formation of an enucleated RBC.[128] Alas, cell reprogramming methodologies at present often disrupt these processes of enucleation and therefore transfusion of in vitro generated RBCs or their immediate precursors the erythroblasts still insufficiently protected against the possibility of tumors formation. However, recently Bouhassira and colleagues found that exposing CD34 positive cells to a short pulse of cytokines favorable for erythroid differentiation prior to stem cell expansion followed by progenitor expansion produced on the order more yield of erythroid cells than the yields observed previously. And the most important: these red blood cells expressed a globin profile similar to that of the developmental age of the CD34 positive cells[129]

See also: Migliaccio AR, Whitsett C, Papayannopoulou T, Sadelain M. (2012) The potential of stem cells as an in vitro source of red blood cells for transfusion. Review. Cell Stem Cell.;10(2):115-9

Platelets [edit]

Platelets play an important role in preventing hemorrhage in thrombocytopenic patients and patients with thrombocythemia. A significant problem for multitransfused patients is refractoriness to platelet transfusions. Thus, the ability to generate platelet products ex vivo and, notably, platelet products lacking HLA antigens in serum free media, would have great clinical value. Some success in this direction has achieved Figueiredo et al. Using an RNA interference-based mechanism in which a lentiviral vector was used to express short-hairpin RNAi targeting β2-microglobulin transcripts in CD34 positive cells, they generated platelets demonstrating an 85% reduction in class I HLA antigens. These platelets appeared to have normal function in vitro[130]

Immune cells [edit]

A specialised type of white blood cell, known as cytotoxic T lymphocytes (CTLs), are produced by the immune system and are able to recognise specific markers on the surface of various infectious or tumour cells, causing them to launch an attack to kill the harmful cells. Thence immunotherapy with functional antigen-specific T cells is potentially an effective therapeutic strategy for combating many types of cancer and viral infection. Unfortunately, sources of such cells for therapeutic purposes are limited, because they are produced in small numbers naturally and have a short lifespan. A potentially efficient approach for generating antigen-specific CTLs is to revert mature immune T cells into iPSCs, which possess indefinite proliferative capacity in vitro, and after their multiplication to coaxed them to redifferentiate back into T cells[131][132][133][134]

Invariant natural killer T (iNKT) cells has great clinical potential as adjuvant for cancer immunotherapy. iNKT cells act as innate T lymphocytes and serve as a bridge between the innate and acquired immune systems. They augment anti-tumor responses by producing interferon-gamma (IFN-γ).[135] A conceptual method is proposed for the use of iPSC-derived iNKT cells for adjuvant cell therapy against cancer, which is composed of four segments: (1) collection of iNKT cells, (2) reprogramming of iNKT cells into iPSCs, (3) re-differentiation of iNKT cell-derived iPSCs into iNKT cells and their expansion in vitro, and (4) injection of iPSC-derived iNKT cells into tumor-bearing animals.[136]

Dendritic cells (DC) are specialized to control T-cell responses. DC with appropriate genetic modifications may survive long enough to stimulate antigen-specific CTL and, after that, be completely eliminated. It was shown that DC-like antigen-presenting cells obtained from human induced pluripotent stem cells may serve as an unlimited source for vaccination therapy.[137]

Induced Mesenchymal stem cells (iMSCs) [edit]

Induction of human iPSc to Generate Mesenchymal Stem/Stromal Cells [edit]

Because of their immunosuppressive properties and ability to differentiate into a wide range of mesenchymal-lineage tissues, mesenchymal stem/stromal cells (MSCs) are under intense investigation for applications in cardiac, renal, neural, joint, and bone repair, as well as in inflammatory conditions and hemopoietic cotransplantation.[138] MSCs are typically harvested from adult bone marrow or fat, but these not only require painful invasive procedures but are low-frequency sources, with MSCs making up only 0.001%– 0.01% of bone marrow cells and 0.05% in liposuction aspirates.[139] Of concern for autologous use, particularly in the elderly most in need of tissue repair, MSCs decline in quantity and quality with age.[138][140] However, induced pluripotent stem cells (iPSCs) could be obtained by the cells rejuvenation of even centenarian human.[8] Because iPSCs can be harvested free of ethical constraints and culture can be expanded indefinitely, they are an advantageous source of MSCs.[141] Chen and colleagues discovered that iPSCs treatment with SB-431542 (that inhibits the activin/TGF- pathways by blocking phosphorylation of ALK4, ALK5, and ALK7 receptors) leads to rapid and uniform MSC generation from human iPSCs. These iPS-MSCs possibly lack teratoma-forming ability, display a normal stable karyotype in culture, and exhibit growth and differentiation characteristics that closely resemble those of primary MSCs described, has considerable potential for the in vitro scale-up required to enable a wide range of MSC-based therapies.[142] Unfortunately, currently there is no data on the in vivo efficacy and long-term safety of iPSC-derived MSCs generated by this method.

It is interesting to note that besides cell therapy in vivo the culture of human mesenchymal stem cells can be used in vitro for mass production of exosomes, which, as it turned out, are ideal vehicles for drug delivery.[143][144][145][146]

Dedifferentiated adipocytes: an alternative source of MSC [edit]

Adipose tissue, because of its abundance and relatively less invasive harvest methods, represents a practical and appealing source of mesenchymal stem cells (MSCs). Unfortunately, there are only 0.05% MSCs in liposuction aspirates.[139] However, a large amount of mature adipocytes, which generally lost their proliferative abilities, and therefore often discarded, can be easily isolated from the adipose cell suspension and dedifferentiated into lipid-free fibroblast-like cells, named dedifferentiated fat (DFAT) cells. DFAT cells re-establish active proliferation ability and undertake multipotent capacities. Compared with adult stem cells, DFAT cells showed unique advantages in their abundance, isolation and homogeneity. Under proper induction culture in vitro or environment in vivo, DFAT cells could demonstrate adipogenic, osteogenic, chondrogenic and myogenic potentials. They also could exhibit perivascular characteristics and elicit neovascularization.[147][148]

Sources of cells for reprogramming [edit]

The most frequently used source for reprogramming are blood cells.[149][150][151][152] and fibroblasts, obtained by biopsy of the skin,[153] but more convenient to receive the body cells from the urine.[154][155][156] This method does not require a biopsy or blood sampling and therefore harmless to the patient.

Another promising source of cells for reprogramming are mesenchymal stem cells derived from human hair follicles.[157]

It is important to note that the origin of somatic cells used for reprogramming may influence the efficiency of reprogramming,[158][159] the functional properties of the resulting induced stem cells[160] and the ability to form tumors.[161][162]

While selecting the source for reprogramming, into account should be taken the fact that iPSCs retain an epigenetic memory of their tissue of origin, and that this impacts their differentiation potential[133][160][163][164][165][166] This epigenetic memory does not necessarily manifest itself at the pluripotency stage – iPSCs derived from different tissues exhibit proper morphology, express pluripotency markers, and are able to differentiate into the three embryonic layers in vitro and in vivo. However, this epigenetic memory may manifest later, during re-differentiation into specific cell types that require the specific loci that have residual epigenetic marks.

Notes [edit]

References for further reading [edit]

References [edit]

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Notes [edit]

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