Stem Cell Clinic

Induced Pluripotent Stem Cells: Problems and Advantages when Applying …

Acta Naturae. 2010 Jul; 2(2): 1828.

Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences

Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences

Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences

Research Center of Clinical and Experimental Medicine, Siberian Branch, Russian Academy of Medical Sciences

Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences

Research Center of Clinical and Experimental Medicine, Siberian Branch, Russian Academy of Medical Sciences

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cells that can be obtained by reprogramming animal and human differentiated cells. In this review, issues related to the nature of iPSCs are discussed and different methods of iPSC production are described. We particularly focused on methods of iPSC production without the genetic modification of the cell genome and with means for increasing the iPSC production efficiency. The possibility and issues related to the safety of iPSC use in cell replacement therapy of human diseases and a study of new medicines are considered.

Keywords: induced pluripotent stem cells, directed stem cell differentiation, cell replacement therapy

Pluripotent stem cells are a unique model for studying a variety of processes that occur in the early development of mammals and a promising tool in cell therapy of human diseases. The unique nature of these cells lies in their capability, when cultured, for unlimited selfrenewal and reproduction of all adult cell types in the course of their differentiation [1]. Pluripotency is supported by a complex system of signaling molecules and gene network that is specific for pluripotent cells. The pivotal position in the hierarchy of genes implicated in the maintenance of pluripotency is occupied by Oct4, Sox2 , and Nanog genes encoding transcription factors [2, 3]. The mutual effect of outer signaling molecules and inner factors leads to the formation of a specific expression pattern, as well as to the epigenome state characteristic of stem cells. Both spontaneous and directed differentiations are associated with changes in the expression pattern and massive epigenetic transformations, leading to transcriptome and epigenome adjustment to a distinct cell type.

Until recently, embryonic stem cells (ESCs) were the only wellstudied source of pluripotent stem cells. ESCs are obtained from either the inner cell mass or epiblast of blastocysts [46]. A series of protocols has been developed for the preparation of various cell derivatives from human ESCs. However, there are constraints for ESC use in cell replacement therapy. The first constraint is the immune incompatibility between the donor cells and the recipient, which can result in the rejection of transplanted cells. The second constraint is ethical, because the embryo dies during the isolation of ESCs. The first problem can be solved by the somatic cell nuclear transfer into the egg cell and then obtaining the embryo and ESCs. The nuclear transfer leads to genome reprogramming, in which ovarian cytoplasmic factors are implicated. This way of preparing pluripotent cells from certain individuals was called therapeutic cloning. However, this method is technologyintensive, and the reprogramming yield is very low. Moreover, this approach encounters the abovementioned ethic problem that, in this case, is associated with the generation of many human ovarian cells [7].

In 2006, the preparation of pluripotent cells by the ectopic expression of four genes Oct4 , Sox2 , Klf4 , and cMyc in both embryonic and adult murine fibroblasts was first reported [8]. The pluripotent cells derived from somatic ones were called induced pluripotent stem cells (iPSCs). Using this set of factors (Oct4, Sox2, Klf4, and cMyc), iPSCs were prepared later from various differentiated mouse [914] and human [1517] cell types. Human iPSCs were obtained with a somewhat altered gene set: Oct4 , Sox2 , Nanog , and Lin28 [18]. Induced PSCs closely resemble ESCs in a broad spectrum of features. They possess similar morphologies and growth manners and are equally sensitive to growth factors and signaling molecules. Like ESCs, iPSCs can differentiate in vitro into derivatives of all three primary germ layers (ectoderm, mesoderm, and endoderm) and form teratomas following their subcutaneous injection into immunodeficient mice. Murine iPSCs injected into blastocysts are normally included in the development to yield animals with a high degree of chimerism. Moreover, murine iPSCs, when injected into tetraploid blastocycts, can develop into a whole organism [19, 20]. Thus, an excellent method that allows the preparation of pluripotent stem cells from various somatic cell types while bypassing ethical problems has been uncovered by researchers.

In the first works on murine and human iPSC production, either retro or lentiviral vectors were used for the delivery of Oct4 , Sox2 , Klf4 , and cMyc genes into somatic cells. The efficiency of transduction with retroviruses is high enough, although it is not the same for different cell types. Retroviral integration into the host genome requires a comparatively high division rate, which is characteristic of the relatively narrow spectrum of cultured cells. Moreover, the transcription of retroviral construct under the control of a promoter localized in 5LTR (long terminal repeat) is terminated when the somatic cell transform switches to the pluripotent state [21]. This feature makes retroviruses attractive in iPSC production. Nevertheless, retroviruses possess some properties that make iPSCs that are produced using them improper for cell therapy of human diseases. First, retroviral DNA is integrated into the host cell genome. The integration occurs randomly; i.e., there are no specific sequences or apparent logic for retroviral integration. The copy number of the exogenous retroviral DNA that is integrated into a genome may vary to a great extent [15]. Retroviruses being integrated into the cell genome can introduce promoter elements and polyadenylation signals; they can also interpose coding sequences, thus affecting transcription. Second, since the transcription level of exogenous Oct4 , Sox2 , Klf4 , and cMyc in the retroviral construct decreases with cell transition into the pluripotent state, this can result in a decrease in the efficiency of the stable iPSC line production, because the switch from the exogenous expression of pluripotency genes to their endogenous expression may not occur. Third, some studies show that the transcription of transgenes can resume in the cells derived from iPSCs [22]. The high probability that the ectopic Oct4 , Sox2 , Klf4 , and cMyc gene expression will resume makes it impossible to apply iPSCs produced with the use of retroviruses in clinical trials; moreover, these iPSCs are hardly applicable even for fundamental studies on reprogramming and pluripotency principles. Lentiviruses used for iPSC production can also be integrated into the genome and maintain their transcriptional activity in pluripotent cells. One way to avoid this situation is to use promoters controlled by exogenous substances added to the culture medium, such as tetracycline and doxycycline, which allows the transgene transcription to be regulated. iPSCs are already being produced using such systems [23].

Another serious problem is the gene set itself that is used for the induction of pluripotency [22]. The ectopic transcription of Oct4 , Sox2 , Klf4 , and cMyc can lead to neoplastic development from cells derived from iPSCs, because the expression of Oct4 , Sox2 , Klf4, and cMyc genes is associated with the development of multiple tumors known in oncogenetics [22, 24]. In particular, the overexpression of Oct4 causes murine epithelial cell dysplasia [25], the aberrant expression of Sox2 causes the development of serrated polyps and mucinous colon carcinomas [26], breast tumors are characterized by elevated expression of Klf4 [27] , and the improper expression of cMyc is observed in 70% of human cancers [28]. Tumor development is oberved in ~50% of murine chimeras obtained through the injection of retroviral iPSCs into blastocysts, which is very likely associated with the reactivation of exogenous cMyc [29, 30].

Several possible strategies exist for resolving the above-mentioned problems:

The search for a less carcinogenic gene set that is necessary and sufficient for reprogramming;

The minimization of the number of genes required for reprogramming and searching for the nongenetic factors facilitating it;

The search for systems allowing the elimination of the exogenous DNA from the host cell genome after the reprogramming;

The development of delivery protocols for nonintegrated genetic constructs;

The search for ways to reprogram somatic cells using recombinant proteins.

The ectopic expression of cMyc and Klf4 genes is the most dangerous because of the high probability that malignant tumors will develop [22]. Hence the necessity to find other genes that could substitute cMyc and Klf4 in iPSC production. It has been reported that these genes can be successfully substituted by Nanog and Lin28 for reprogramming human somatic cells [18;] . iPSCs were prepared from murine embryonic fibroblasts by the overexpression of Oct4 and Sox2 , as well as the Esrrb gene encoding the murine orphan nuclear receptor beta. It has already been shown that Esrrb , which acts as a transcription activator of Oct4 , Sox2 , and Nanog , is necessary for the selfrenewal and maintenance of the pluripotency of murine ESCs. Moreover, Esrrb can exert a positive control over Klf4 . Thus, the genes causing elevated carcinogenicity of both iPSCs and their derivatives can be successfully replaced with less dangerous ones [31].

The Most Effectively Reprogrammed Cell Lines . Murine and human iPSCs can be obtained from fibroblasts using the factors Oct4, Sox2, and Klf4, but without cMyc . However, in this case, reprogramming decelerates and an essential shortcoming of stable iPSC clones is observed [32, 33]. The reduction of a number of necessary factors without any decrease in efficiency is possible when iPSCs are produced from murine and human neural stem cells (NSCs) [12, 34, 35]. For instance, iPSCs were produced from NSCs isolated from adult murine brain using two factors, Oct4 and Klf4, as well as even Oct4 by itself [12, 34]. Later, human iPSCs were produced by the reprogramming of fetal NSCs transduced with a retroviral vector only carrying Oct4 [35] . It is most likely that the irrelevance of Sox2, Klf4, and cMyc is due to the high endogenous expression level of these genes in NSCs.

Successful reprogramming was also achieved in experiments with other cell lines, in particular, melanocytes of neuroectodermal genesis [36]. Both murine and human melanocytes are characterized by a considerable expression level of the Sox2 gene, especially at early passages. iPSCs from murine and human melanocytes were produced without the use of Sox2 or cMyc. However, the yield of iPSC clones produced from murine melanocytes was lower (0.03% without Sox2 and 0.02% without cMyc) in comparison with that achieved when all four factors were applied to melanocytes (0.19%) and fibroblasts (0.056%). A decreased efficiency without Sox2 or cMyc was observed in human melanocyte reprogramming (0.05% with all four factors and 0.01% without either Sox2 or cMyc ). All attempts to obtain stable iPSC clones in the absence of both Sox2 and cMyc were unsuccessful [36]. Thus, the minimization of the number of factors required for iPSC preparation can be achieved by choosing the proper somatic cell type that most effectively undergoes reprogramming under the action of fewer factors, for example, due to the endogenous expression of pluripotency genes. However, if human iPSCs are necessary, these somatic cells should be easily accessible and wellcultured and their method of isolation should be as noninvasive as possible.

One of these cell types can be adipose stem cells (ASCs). This is a heterogeneous group of multipotent cells which can be relatively easily isolated in large amounts from adipose tissue following liposuction. Human iPSCs were successfully produced from ASCs with a twofold reprogramming rate and 20fold efficiency (0.2%), exceeding those of fibroblasts [37].

However, more accessible resources for the effective production of human iPSCs are keratinocytes. When compared with fibroblasts, human iPSC production from keratinocytes demonstrated a 100fold greater efficiency and a twofold higher reprogramming rate [38].

It has recently been found that the reprogramming of murine papillary dermal fibroblasts (PDFs) into iPSCs can be highly effective with the overexpression of only two genes, Oct4 and Klf4 , inserted into retroviral vectors [39;]. PDFs are specialized cells of mesodermal genesis surrounding the stem cells of hair follicles . One characteristic feature of these cells is the endogenous expression of Sox2 , Klf4 , and cMyc genes, as well as the geneencoding alkaline phosphatase, one of the murine and human ESC markers. PDFs can be easily separated from other cell types by FACS (fluorescenceactivated cell sorting) using life staining with antibodies against the surface antigens characteristic of one or another cell type. The PDF reprogramming efficiency with the use of four factors (Oct4, Sox2, Klf4, and cMyc) retroviral vectors is 1.38%, which is 1,000fold higher than the skin fibroblast reprogramming efficiency in the same system. Reprogramming PDFs with two factors, Oct4 and Klf4 , yields 0.024%, which is comparable to the efficiency of skin fibroblast reprogramming using all four factors. The efficiency of PDF reprogramming is comparable with that of NSCs, but PDF isolation is steady and far less invasive [39]. It seems likely that human PDF lines are also usable, and this cell type may appear to be one of the most promising for human iPSC production in terms of pharmacological studies and cell replacement therapy. The use of such cell types undergoing more effective reprogramming, together with methods providing the delivery of pluripotency genes without the integration of foreign DNA into the host genome and chemical compounds increasing the reprogramming efficiency and substituting some factors required for reprogramming, is particularly relevant.

Chemical Compounds Increasing Cell Reprogramming Efficiency. As was noted above, the minimization of the factors used for reprogramming decreases the efficiency of iPSC production. Nonetheless, several recent studies have shown that the use of genetic mechanisms, namely, the initiation of ectopic gene expression, can be substituted by chemical compounds, most of them operating at the epigenetic level. For instance, BIX01294 inhibiting histone methyltransferase G9a allows murine fibroblast reprogramming using only two factors, Oct4 and Klf4, with a fivefold increased yield of iPSC clones in comparison with the control experiment without BIX01294 [40]. BIX01294 taken in combination with another compound can increase the reprogramming efficiency even more. In particular, BIX01294 plus BayK8644 elevated the yield of iPCSs 15 times, and BIX01294 plus RG108 elevated it 30 times when only two reprogramming factors, Oct4 and Klf4, were used. RG108 is an inhibitor of DNA methyltransferases, and its role in reprogramming is apparently in initiating the more rapid and effective demethylation of promoters of pluripotent cellspecific genes, whereas BayK8644 is an antagonist of Ltype calcium channels, and its role in reprogramming is not understood very well [40]. However, more considerable results were obtained in reprogramming murine NSCs. The use of BIX01294 allowed a 1.5fold increase in iPSC production efficiency with two factors, Oct4 and Klf4, in comparison with reprogramming with all four factors. Moreover, BIX01294 can even substitute Oct4 in the reprogramming of NSCs, although the yield is very low [41]. Valproic (2propylvaleric) acid inhibiting histone deacetylases can also substitute cMyc in reprogramming murine and human fibroblasts. Valproic acid (VPA) increases the reprogramming efficiency of murine fibroblasts 50 times, and human fibroblasts increases it 1020 times when three factors are used [42, 43]. Other deacetylase inhibitors, such as TSA (trichostatin A) and SAHA (suberoylanilide hyroxamic acid), also increase the reprogramming efficiency. TSA increases the murine fibroblast reprogramming efficiency 15 times, and SAHA doubles it when all four factors are used [42]. Besides epigenetic regulators, the substances inhibiting the protein components of signaling pathways implicated in the differentiation of pluripotent cells are also applicable in the substitution of reprogramming factors. In particular, inhibitors of MEK and GSK3 kinases (PD0325901 and CHIR99021, respectively) benefit the establishment of the complete and stable pluripotency of iPSCs produced from murine NSCs using two factors, Oct4 and Klf4 [41, 44].

It has recently been shown that antioxidants can considerably increase the efficiency of somatic cell reprogramming. Ascorbic acid (vitamin C) can essentially influence the efficiency of iPSC production from various murine and human somatic cell types [45]. The transduction of murine embryonic fibroblasts (mEFs) with retroviruses carrying the Oct4 , Sox2 , and Klf4 genes results in a significant increase in the production level of reactive oxygen species (ROS) compared with that of both control and Efs tranduced with Oct4 , Sox2 , cMyc , and Klf4 . In turn, the increase in the ROS level causes accelerated aging and apoptosis of the cell, which should influence the efficiency of cell reprogramming. By testing several substances possessing antioxidant activity such as vitamin B1, sodium selenite, reduced glutathione, and ascorbic acid, the authors have found that combining these substances increases the yield of GFPpositive cells in EF reprogramming (the Gfp gene was under the control of the Oct4 gene promoter). The use of individual substances has shown that only ascorbate possesses a pronounced capability to increase the level of GFPpositive cells, although other substances keep their ROSdecreasing ability. In all likelihood, this feature of ascorbates is not directly associated with its antioxidant activity [45]. The score of GFPpositive iPSC colonies expressing an alkaline phosphatase has shown that the efficiency of iPSC production from mEFs with three factors (Oct4, Sox2, and Klf4) can reach 3.8% in the presence of ascorbate. When all four factors (Oct4, Sox2, Klf4, and cMyc) are used together with ascorbate, the efficiency of iPSC production may reach 8.75%. A similar increase in the iPSC yield was also observed in the reprogramming of murine breast fibroblasts; i.e., the effect of vitamin C is not limited by one cell type. Moreover, the effect of vitamin C on the reprogramming efficiency is more profound than that of the deacetylase inhibitor valproic (2propylvaleric) acid. The mutual effect of ascorbate and valproate is additive; i.e., these substances have different action mechanisms. Moreover, vitamin C facilitates the transition from preiPSCs to stable pluripotent cells. This feature is akin to the effects of PD0325901 and CHIR99021, which are inhibitors of MEK and GSK3 kinases, respectively. This effect of vitamin C expands to human cells as well [45]. Following the transduction of human fibroblasts with retroviruses carrying Oct4 , Sox2 , Klf4 , and cMyc and treatment with ascorbate, the authors prepared iPSCs with efficiencies reaching 6.2%. The reprogramming efficiency of ASCs under the same conditions reached 7.06%. The mechanism of the effect that vitamin C has on the reprogramming efficiency is not known in detail. Nevertheless, the acceleration of cell proliferation was observed at the transitional stage of reprogramming. The levels of the p53 and p21 proteins decreased in cells treated with ascorbate, whereas the DNA repair machinery worked properly [45]. It is interesting that an essential decrease in the efficiency of iPSC production has been shown under the action of processes initiated by p53 and p21 [4650].

As was mentioned above, for murine and human iPSC production, both retro and lentiviruses were initially used as delivery vectors for the genes required for cell reprogramming. The main drawback of this method is the uncontrolled integration of viral DNA into the host cells genome. Several research groups have introduced methods for delivering pluripotency genes into the recipient cell which either do not integrate allogenic DNA into the host genome or eliminate exogenous genetic constructs from the genome.

CreloxP Mediated Recombination. To prepare iPSCs from patients with Parkinsons disease, lentiviruses were used, the proviruses of which can be removed from the genome by Cre recombinase. To do this, the loxP site was introduced into the lentiviral 3LTRregions containing separate reprogramming genes under the control of the doxycyclineinducible promoter. During viral replication, loxP was duplicated in the 5LTR of the vector. As a result, the provirus integrated into the genome was flanked with two loxP sites. The inserts were eliminated using the temporary transfection of iPSCs with a vector expressing Cre recombinase [51].

In another study, murine iPSCs were produced using a plasmid carrying the Oct4 , Sox2 , Klf4I, and cMyc genes in the same reading frame in which individual cDNAs were separated by sequences encoding 2 peptides, and practically the whole construct was flanked with loxP sites [52]. The use of this vector allowed a notable decrease in the number of exogenous DNA inserts in the host cells genome and, hence, the simplification of their following excision [52]. It has been shown using lentiviruses carrying similar polycistronic constructs that one copy of transgene providing a high expression level of the exogenous factors Oct4, Sox2, Klf4, and cMyc is sufficient for the reprogramming of differentiated cells into the pluripotent state [53, 54].

The drawback of the CreloxP system is the incomplete excision of integrated sequences; at least the loxP site remains in the genome, so the risk of insertion mutations remains.

Plasmid Vectors . The application of lentiviruses and plasmids carrying the loxP sites required for the elimination of transgene constructs modifies, although insignificantly, the host cells genome. One way to avoid this is to use vector systems that generally do not provide for the integration of the whole vector or parts of it into the cells genome. One such system providing a temporary transfection with polycistronic plasmid vectors was used for iPSC production from mEFs [29]. A polycistronic plasmid carrying the Oct4 , Sox2 , and Klf4 gene cDNAs, as well as a plasmid expressing cMyc , was transfected into mEFs one, three, five, and seven days after their primary seeding. Fibroblasts were passaged on the ninth day, and the iPSC colonies were selected on the 25th day. Seven out of ten experiments succeeded in producing GFPpositive colonies (the Gfp gene was under the control of the Nanog gene promoter). The iPSCs that were obtained were similar in their features to murine ESCs and did not contain inserts of the used DNA constructs in their genomes. Therefore, it was shown that wholesome murine iPSCs that do not carry transgenes can be reproducibly produced, and that the temporary overexpression of Oct4 , Sox2 , Klf4 , and cMyc is sufficient for reprogramming. The main drawback of this method is its low yield. In ten experiments the yield varied from 1 to 29 iPSC colonies per ten million fibroblasts, whereas up to 1,000 colonies per ten millions were obtained in the same study using retroviral constructs [29].

Episomal Vectors . Human iPSCs were successfully produced from skin fibroblasts using single transfection with polycistronic episomal constructs carrying various combinations of Oct4 , Sox2 , Nanog , Klf4 , cMyc , Lin28 , and SV40LT genes. These constructs were designed on the basis of the oriP/EBNA1 (EpsteinBarr nuclear antigen1) vector [55]. The oriP/EBNA1 vector contains the IRES2 linker sequence allowing the expression of several individual cDNAs (encoding the genes required for successful reprogramming in this case) into one polycistronic mRNA from which several proteins are translated. The oriP/EBNA1 vector is also characterized by lowcopy representation in the cells of primates and can be replicated once per cell cycle (hence, it is not rapidly eliminated, the way common plasmids are). Under nonselective conditions, the plasmid is eliminated at a rate of about 5% per cell cycle [56]. In this work, the broad spectrum of the reprogramming factor combinations was tested, resulting in the best reprogramming efficiency with cotransfection with three episomes containing the following gene sets: Oct4 + Sox2 + Nanog + Klf4 , Oct4 + Sox2 + SV40LT + Klf4 , and cMyc + Lin28 . SV40LT ( SV40 large T gene ) neutralizes the possible toxic effect of overexpression [57]. The authors have shown that wholesome iPSCs possessing all features of pluripotent cells can be produced following the temporary expression of a certain gene combination in human somatic cells without the integration of episomal DNA into the genome. However, as in the case when plasmid vectors are being used, this way of reprogramming is characterized by low efficiency. In separate experiments the authors obtained from 3 to 6 stable iPSC colonies per 106 transfected fibroblasts [55]. Despite the fact that skin fibroblasts are wellcultured and accessible, the search for other cell types which are relatively better cultured and more effectively subject themselves to reprogramming through this method is very likely required. Another drawback of the given system is that this type of episome is unequally maintained in different cell types.

PiggyBacTransposition . One promising system used for iPSC production without any modification of the host genome is based on DNA transposons. Socalled PiggyBac transposons containing 2linkered reprogramming genes localized between the 5 and 3terminal repeats were used for iPSC production from fibroblasts. The integration of the given constructs into the genome occurs due to mutual transfection with a plasmid encoding transposase. Following reprogramming due to the temporary expression of transposase, the elimination of inserts from the genome took place [58, 59]. One advantage of the PiggyBac system on CreloxP is that the exogenous DNA is completely removed [60].

However, despite the relatively high efficiency of exogenous DNA excision from the genome by PiggyBac transposition, the removal of a large number of transposon copies is hardly achievable.

Nonintegrating Viral Vectors . Murine iPSCs were successfully produced from hepatocytes and fibroblasts using four adenoviral vectors nonintegrating into the genome and carrying the Oct4 , Sox2 , Klf4 , and cMyc genes. An analysis of the obtained iPSCs has shown that they are similar to murine ESCs in their properties (teratoma formation, gene promoter DNA methylation, and the expression of pluripotent markers), but they do not carry insertions of viral DNA in their genomes [61]. Later, human fibroblastderived iPSCs were produced using this method [62].

The authors of this paper cited the postulate that the use of adenoviral vectors allows the production of iPSCs, which are suitable for use without the risk of viral or oncogenic activity. Its very low yield (0.00010.001%), the deceleration of reprogramming, and the probability of tetraploid cell formation are the drawbacks of the method. Not all cell types are equally sensitive to transduction with adenoviruses.

Another method of gene delivery based on viral vectors was recently employed for the production of human iPSCs. The sendaivirus (SeV)based vector was used in this case [63]. SeV is a singlestranded RNA virus which does not modify the genome of recipient cells; it seems to be a good vector for the expression of reprogramming factors. Vectors containing either all pluripotency factors or three of them (without ) were used for reprogramming the human fibroblast. The construct based on SeV is eliminated later in the course of cell proliferation. It is possible to remove cells with the integrated provirus via negative selection against the surface HN antigen exposed on the infected cells. The authors postulate that reprogramming technology based on SeV will enable the production of clinically applicable human iPSCs [63].

Cell Transduction with Recombinant Proteins . Although the methods for iPSC production without gene modification of the cells genome (adenoviral vectors, plasmid gene transfer, etc.) are elaborated, the theoretical possibility for exogenous DNA integration into the host cells genome still exists. The mutagenic potential of the substances used presently for enhancing iPSC production efficiency has not been studied in detail. Fully checking iPSC genomes for exogenous DNA inserts and other mutations is a difficult task, which becomes impossible to solve in bulk culturing of multiple lines. The use of protein factors delivered into a differentiated cell instead of exogenous DNA may solve this problem. Two reports have been published to date in which murine and human iPSCs were produced using the recombinant Oct4, Sox2, Klf4, and cMyc proteins [64, 65] . T he method used to deliver the protein into the cell is based on the ability of peptides enriched with basic residues (such as arginine and lysine) to penetrate the cells membrane. Murine iPSCs were produced using the recombinant Oct4, Sox2, Klf4, and cMyc proteins containing eleven Cterminal arginine residues and expressed in E. coli . The authors succeeded in producing murine iPSCs during four rounds of protein transduction into embryonic fibroblasts [65]. However, iPSCs were only produced when the cells were additionally treated with 2propylvalerate (the deacetylase inhibitor). The same principle was used for the production of human iPSCs, but protein expression was carried out in human HEK293 cells, and the proteins were expressed with a fragment of nine arginins at the protein Cend. Researchers have succeeded in producing human iPSCs after six transduction rounds without any additional treatment [64]. The efficiency of producing human iPSC in this way was 0.001%, which is one order lower than the reprogramming efficiency with retroviruses. Despite some drawbacks, this method is very promising for the production of patientspecific iPSCs.

The first lines of human pluripotent ESCs were produced in 1998 [6]. In line with the obvious fundamental importance of embryonic stem cell studies with regard to the multiple processes taking place in early embryogenesis, much of the interest of investigators is associated with the possibility of using ESCs and their derivatives as models for the pathogenesis of human diseases, new drugs testing, and cell replacement therapy. Substantial progress is being achieved in studies on directed human ESC differentiation and the possibility of using them to correct degenerative disorders. Functional cell types, such as motor dopaminergic neurons, cardiomyocytes, and hematopoietic cell progenitors, can be produced as a result of ESC differentiation. These cell derivatives, judging from their biochemical and physiological properties, are potentially applicable for the therapy of cardiovascular disorders, nervous system diseases, and human hematological disorders [66]. Moreover, derivatives produced from ESCs have been successfully used for treating diseases modeled on animals. Therefore, bloodcell progenitors produced from ESCs were successfully used for correcting immune deficiency in mice. Visual functions were restored in blind mice using photoreceptors produced from human ESCs, and the normal functioning of the nervous system was restored in rats modeling Parkinsons disease using the dopaminergic neurons produced from human ESCs [6770]. Despite obvious success, the fullscale application of ESCs in therapy and the modeling of disorders still carry difficulties, because of the necessity to create ESC banks corresponding to all HLAhaplotypes, which is practically unrealistic and hindered by technical and ethical problems.

Induced pluripotent stem cells can become an alternative for ESCs in the area of clinical application of cell replacement therapy and screening for new pharmaceuticals. iPSCs closely resemble ESCs and, at the same time, can be produced in almost unlimited amounts from the differentiated cells of each patient. Despite the fact that the first iPSCs were produced relatively recently, work on directed iPSC differentiation and the production of patientspecific iPSCs is intensive, and progress in this field is obvious.

Dopamine and motor neurons were produced from human iPSCs by directed differentiation in vitro [71, 72]. These types of neurons are damaged in many inherited or acquired human diseases, such as spinal cord injury, Parkinsons disease, spinal muscular atrophy, and amyotrophic lateral sclerosis. Some investigators have succeeded in producing various retinal cells from murine and human iPSCs [7375]. Human iPSCs have been shown to be spontaneously differentiated in vitro into the cells of retinal pigment epithelium [76]. Another group of investigators has demonstrated that treating human and murine iPSCs with Wnt and Nodal antagonists in a suspended culture induces the appearance of markers of cell progenitors and pigment epithelium cells. Further treating the cells with retinoic acid and taurine activates the appearance of cells expressing photoreceptor markers [75].

Several research groups have produced functional cardiomyocytes (CMs) in vitro from murine and human iPSCs [7781]. Cardiomyocytes produced from iPSC are very similar in characteristics (morphology, marker expression, electrophysiological features, and sensitivity to chemicals) to the CMs of cardiac muscle and to CMs produced from differentiated ESCs. Moreover, murine iPSCs, when injected, can repair muscle and endothelial cardiac tissues damaged by cardiac infarction [77].

Hepatocytelike cell derivatives, dendritic cells, macrophages, insulinproducing cell clusters similar to the duodenal islets of Langerhans, and hematopoietic and endothelial cells are currently produced from murine and human iPSCs, in addition to the alreadylisted types of differentiated cells [8285].

In addition to directed differentiation in vitro , investigators apply much effort at producing patientspecific iPSCs. The availability of pluripotent cells from individual patients makes it possible to study pathogenesis and carry out experiments on the therapy of inherited diseases, the development of which is associated with distinct cell types that are hard to obtain by biopsy: so the use of iPSCs provides almost an unlimited resource for these investigations. Recently, the possibility of treating diseases using iPSCs was successfully demonstrated, and the design of the experiment is presented in the figure. A mutant allele was substituted with a normal allele via homologous recombination in murine fibroblasts representing a model of human sickle cell anemia. iPSCs were produced from repaired fibroblasts and then differentiated into hematopoietic cell precursors. The hematopoietic precursors were then injected into a mouse from which the skin fibroblasts were initially isolated (). As a result, the initial pathological phenotype was substantially corrected [86]. A similar approach was applied to the fibroblasts and keratinocytes of a patient with Fanconis anemia. The normal allele of the mutant gene producing anemia was introduced into a somatic cell genome using a lentivirus, and then iPSCs were obtained from these cells. iPSCs carrying the normal allele were differentiated into hematopoietic cells maintaining a normal phenotype [87]. The use of lentiviruses is unambiguously impossible when producing cells to be introduced into the human body due to their oncogenic potential. However, new relatively safe methods of genome manipulation are currently being developed; for instance, the use of synthetic nucleases containing zinc finger domains allowing the effective correction of genetic defects in vitro [88].

Design of an experiment on repairing the mutant phenotype in mice modeling sickle cell anemia development [2]. Fibroblasts isolated from the tail of a mouse (1) carrying a mutant allele of the gene encoding the human hemoglobin -chain (hs) were used for iPSC production (2). The mutation was then repaired in iPSCs by means of homological recombination (3) followed by cell differentiation via the embryoid body formation (4). The directed differentiation of the embryoid body cells led to hematopoietic precursor cells (5) that were subsequently introduced into a mouse exposed to ionizing radiation (6).

The induced pluripotent stem cells are an excellent model for pathogenetic studies at the cell level and testing compounds possessing a possible therapeutic effect.

The induced pluripotent stem cells were produced from the fibroblasts of a patient with spinal muscular atrophy (SMA) (SMAiPSCs). SMA is an autosomal recessive disease caused by a mutation in the SMN1 ( survival motor neuron 1 ) gene, which is manifested as the selective nonviability of lower motor neurons. Patients with this disorder usually die at the age of about two years. Existing experimental models of this disorder based on the use of flatworms, drosophila, and mice are not satisfactory. The available fibroblast lines from patients with SMA cannot provide the necessary data on the pathogenesis of this disorder either. It was shown that motor neurons produced from SMAiPSCs can retain the features of SMA development, selective neuronal death, and the lack of SMN1 transcription. Moreover, the authors succeeded in elevating the SMN protein level and aggregation (encoded by the SMN2 gene, whose expression can compensate for the shortage in the SMN1 protein) in response to the treatment of motor neurons and astrocytes produced from SMAiPSCs with valproate and torbomycin [89;]. iPSCs and their derivatives can serve as objects for pharmacological studies, as has been demonstrated on iPSCs from patients with familial dysautonomia (FDA) [90]. FDA is an inherited autosomal recessive disorder manifested as the degeneration of sensor and autonomous neurons. This is due to a mutation causing the tissuespecific splicing of the IKBKAP gene, resulting in a decrease in the level of the fulllength IKAP protein. iPSCs were produced from fibroblasts of patients with FDA. They possessed all features of pluripotent cells. Neural derivatives produced from these cells had signs of FDA pathogenesis and low levels of the fulllength IKBKAP transcript. The authors studied the effect of three substances, kinetin, epigallocatechin gallate, and tocotrienol, on the parameters associated with FDA pathogenesis. Only kinetin has been shown to induce an increase in the level of fulllength IKBKAP transcript. Prolonged treatment with kinetin induces an increase in the level of neuronal differentiation and expression of peripheral neuronal markers.

Currently, a broad spectrum of iPSCs is produced from patients with various inherited pathologies and multifactorial disorders, such as Parkinsons disease, Down syndrome, type 1 diabetes, Duchenne muscular dystrophy, talassemia, etc., which are often lethal and can scarcely be treated with routine therapy [51, 87, 89, 9194]. The data on iPSCs produced by reprogramming somatic cells from patients with various pathologies are given in the .

Functional categories of M. tuberculosis genes with changed expression level during transition to the NC state

One can confidently state that both iPSCs themselves and their derivatives are potent instruments applicable in biomedicine, cell replacement therapy, pharmacology, and toxicology. However, the safe application of iPSCbased technologies requires the use of methods of iPSCs production and their directed differentiation which minimize both the possibility of mutations in cell genomes under in vitro culturing and the probability of malignant transformation of the injected cells. The development of methods for human iPSC culturing without the use of animal cells (for instance, the feeder layer of murine fibroblasts) is necessary; they make a viralorigin pathogen transfer from animals to humans impossible. There is a need for the maximum standardization of conditions for cell culturing and differentiation.

This study was supported by the Russian Academy of Sciences Presidium Program Molecular and Cell Biology.

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Induced Pluripotent Stem Cells: Problems and Advantages when Applying ...

Induced Pluripotent Stem Cell (iPSC)Derived Lymphocytes for Adoptive …

Curr Hematol Malig Rep. 2019; 14(4): 261268.

Department of Hematology, Amsterdam University Medical Centers, Cancer Center Amsterdam, Location VUmc, Amsterdam, Netherlands

Department of Hematology, Amsterdam University Medical Centers, Cancer Center Amsterdam, Location VUmc, Amsterdam, Netherlands

Department of Hematology, Amsterdam University Medical Centers, Cancer Center Amsterdam, Location VUmc, Amsterdam, Netherlands

In the rapidly developing field of adoptive cell immunotherapy, there is urgent need for discoveries that would improve outcomes, extend the applicability, and reduce the costs. Induced pluripotent stem cells (iPSC) can be a source of broadly applicable cellular immunotherapeutics, which have been manufactured, validated, and banked in advance, and can be applied across HLA barriers. Here, we discuss the recent advances and challenges in the generation of iPSC-derived cellular products for cancer therapy.

iPSCs can be differentiated to functional tumor-specific T and NK cells in vitro with demonstrable in vitro and in vivo anti-tumor activity. Genetic modifications employed at the iPSC level can deliver desirable immunotherapeutic attributes to the generated immune effectors. iPSC-NK cells are currently evaluated in a clinical setting and pre-clinical testing of iPSC-T cells shows promising results but their production seems more challenging.

The use of iPSCs for the generation of tumor-targeting T/NK cells constitutes a feasible strategy to overcome limitations in manufacturing, efficacy, and applicability of cellular therapeutics.

Keywords: Induced pluripotent stem cells, Adoptive cell immunotherapy, NK cells, T cells, Off-the-shelf

Adoptive cell immunotherapy for the treatment of hematological malignancies has greatly advanced in the last decades, leading to significant clinical outcomes. Both T cells and NK cells have been proven robust therapeutic agents and several approaches to obtain anti-tumor therapeutic lymphocytes have been proposed with variable clinical impact. These include the isolation and expansion of patient-derived tumor antigen-specific T cells (T cell clones, tumor infiltrating lymphocytes), innate T cells (NK-T or -T cells), or NK cells [1, 2]. The introduction of genetic engineering with T cell receptors (TCR) and most importantly chimeric antigen receptors (CAR) boosted the applicability and efficacy of adoptive cell immunotherapy [3]. CARs are artificial receptors that redirect antigen recognition from T cells and mediate T cell activation, through the fusion of an extracellular antigen-binding moiety, such as a single-chain-variable region (scFv), with an intracellular signaling domain [4]. CAR-modified T cells (CAR-T) targeting CD19 have induced impressive responses in chemotherapy-resistant B cell leukemias and lymphomas [5, 6], while B cell maturation antigen (BCMA)targeting CAR-T cells show promising clinical results against multiple myeloma [7]. These remarkable results led further to the development of CAR-engineered NK cells (CAR-NK), redirecting the intrinsic capacity of NK cells for tumor recognition and elimination and the initiation of CAR-NK clinical trials [8, 9].

Although successful, current approaches of adoptive cell therapy have significant limitations that impede their further progress and broader use. Immunotherapy using primary T cells is mainly performed in an autologous setting limiting a facile and general use. The production of genetically engineered therapeutic cells is a time-consuming process and the required processing time can be detrimental for the patients health. Also, in many cases, the autologous T cell isolation and expansion could be problematic and their functionality and quality doubtful (e.g., patients receiving highly immunosuppressive therapy). In addition, the existing ex vivo T cell expansion protocols push T cells to a terminal differentiated effector state, resulting in exhausted, less effective cellular products. The use of allogeneic cells from volunteer donors has the potential to broaden the applicability of the T cell products to HLA-matched recipients [10]. The advances of gene editing technology allow for further broadening of the applicability of CAR-T cell therapy by the use of allogeneic volunteer donor T cells that have been modified to lack TCR expression and thus, avoid graft-versus-host disease (GvHD) [10]. However, the final purity of the TCR-less CAR-T cell product is not always acceptable. Carriage of even <1% (<45104 cells/kg) of residual TCR-expressing T cells was still enough to initiate GvHD in the first clinical application of TCR-less CAR-T cells [10]. In addition, the potential off-target genetic alterations require further safety testing of every different batch produced that brings higher production costs. Extra gene editing to also eliminate HLA class I expression and reduce the risk of rejection could further reduce production efficiency (60% of double targeting efficiency) [10, 11] and require extra ex vivo expansion in order to reach the desirable yield of fully edited cells. However, it is known that longer ex vivo culture can be at the cost of having a more exhausted final product [12]. Notably, NK cells exert their function regardless of the recipients HLA haplotype and thus they can be isolated from an unrelated donor or cord blood. Clinical trials using adoptively transferred allogeneic NK cells showed limited toxic side effects such as GvHD [1317]. However, additionally to the above-mentioned limitations, their inadequate proliferative capacity impedes their in vivo persistence without cytokine support [18] and makes multiplex gene editing challenging. Immortalized NK cell lines, such as NK92, have been used as an alternative but they have to be irradiated before being infused to the patient [9, 19]. This limits their in vivo survival and thus, multiple doses of high numbers of NK92 cells are required [9]. Clinical application of irradiated NK92 cells transduced with a CD33-CAR was safe but without impressive anti-tumor results [9].

The development of broadly applicable cellular therapeutics, which have been manufactured, functionally validated and banked in advance, and can be applied across HLA barriers would improve the consistency and availability and reduce the cost of adoptive cell therapy. The generation of human lymphocytes from iPSCs has attracted lately the interest of the scientific community since it offers tantalizing prospects for cell-based therapies serving as an endless supply of custom-made, off-the-shelf therapeutic lymphocytes. Here, we review the latest advances in generating therapeutic anti-tumor T and NK lymphocytes from human induced pluripotent stem cells (iPSCs) and future challenges towards the final development of universally applicable immunotherapeutic products.

The iPSC technology offers new perspectives for the production of immunotherapeutic cellular products due to two major characteristics. First, similar to embryonic stem cells, iPSCs can be cultured unlimitedly in vitro and be successfully differentiated towards the lymphoid lineage [20]. Having access to constant and continuous production of T and/or NK lymphocytes offers solution to cell number and doses limitations due to restricted availability or expansion of primary cells. Second, iPSCs can be easily amenable to genetic transformations in vitro and thus, can generate immune effectors, which may eventually be genetically modified to augment their applicability, potency, and persistence. While the potential for multiplex gene editing is limited in primary cells, iPSC can theoretically bear unlimited genetic changes. Finally, in contrast to primary cells [10], genetic engineering of iPSCs results in fully modified clonal lines, which could be extensively evaluated resulting in a stable and safe source. The generation of safe master iPSC lines, bearing genetic modifications that confer the desired characteristics to the final product, would facilitate the development of off-the-shelf cellular therapeutics for more patients and types of malignancy.

Up to date almost all somatic cell types have been successfully reprogrammed into iPSCs through introduction of defined transcription factors (Oct4, Sox2, Klf4, c-Myc or Oct4, Sox2, Nanog, Lin28) [21, 22]. However, the selection of the initial iPSC source seems to be important when aiming in the efficient generation of therapeutically relevant T or NK lymphocytes (Fig.). Although, iPSC, from any somatic cell, can be successfully differentiated towards the lymphoid lineage, it has been shown that starting from blood cell-derived iPSCs, such as CD34+ cells from cord blood, monocytes or peripheral blood lymphocytes, results in a more efficient generation of CD4+CD8+ double-positive (DP) thymocytes in vitro [23] suggesting a level of epigenetic memory [24]. Moreover, iPSCs from peripheral blood T lymphocytes (T-iPSC) have the unique characteristic of bearing the rearranged TCR loci of the parental cells, which remain unchanged during in vitro differentiation [21, 23]. Therefore, T cells with defined TCR-specificity (e.g., T cell clones, invariant T cells, NK-T cells) can be selected for reprogramming to T-iPSC for therapeutic T cell production.

a Peripheral blood cells serve as a primary source to generate iPSC lines by non-integrating delivery of reprogramming transcription factors. b Generation of iPSC-derived off-the-shelf tumor-specific T cells. iPSCs are genetically modified to bear desirable immunotherapeutic properties. The expression of TCR and HLA is knocked out or silenced to prevent alloreactivity and graft rejection respectively. HLA-E/G molecules can be overexpressed to avoid NK cellmediated transplant rejection, whereas antigen-specific TCR/CARs can direct anti-tumor activity. Further, introduction of the expression of immune receptors, cytokines, chemokines, or other immune regulatory factors may enhance anti-tumor function. Genome-edited master iPSC lines are differentiated under GMP-grade conditions to fully functional histocompatible tumor-targeting T cells accessible to all patients regardless of their HLA haplotype

The hallmark of adoptive T cell immunotherapy is the use of the ability of T cells to specifically recognize tumor antigens. Naturally, this ability is endowed through the expression of specific TCRs encoded by the uniquely rearranged genomic loci of the TCR and chains. Generation of T cells from embryonic stem cells or iPSC, which bear non-rearranged germline TCR loci, results in random rearrangements during differentiation and a population of cells with various unknown specificities [25]. Nishimura et al. demonstrated that differentiation of T-iPSCderived from an antigen-specific T cell clone gives rise to T cells with the same TCR rearrangement and reactivity [21], thus making T-iPSC from tumor antigen-specific T cells a way to deliver defined specificity to iPSC-derived T cells. Until now, T cell clones specific for several tumor antigens, such as MART-1 (melanoma), LMP2 (EBV antigen), WT-1 (leukemia), and GPC3 (hepatocellular, ovarian, and lung carcinoma), have been reprogrammed to T-iPSC [23, 26, 27]. Importantly, tumor-specific T-iPSC could give rise to cytotoxic CD8 single-positive (SP) T cells, which could recognize the target antigen on cell lines and display specific cytotoxicity [23, 26, 27]. Although most of the generated cells bear the original tumor-specific TCR, it has been observed that TCR can be additionally rearranged, resulting in TCR destabilization and loss of the antigen specificity [26]. Inactivation of recombination activating gene 2 (RAG2) in the T-iPSC, a key protein complex in the rearrangement of TCR, can inhibit the process of further TCR rearrangement at the DP stage and result in preservation of antigen specificity in CD8 T cells [26].

Although taking advantage of a naturally occurring TCR in order to convey antigen specificity to iPSC-derived T cells, one can imagine that the flexibility of this method is limited, as the existence of pre-made, HLA-matched antigen-specific T cell clones is a requirement. The advances in engineering of immune cells open up new perspectives for the generation of custom-made synthetic T cells from iPSC. Antigen specificity can be assigned to iPSCs and their T cell derivatives by means of a transgenic TCR [26]. Themeli et al. demonstrated the feasibility of generating functional CAR-T cells by engineering T-iPSC with a CAR [28]. The use of CARs endows T-iPSCderived T cells with HLA-independent, customizable antigen recognition as scFv domains of different specificity can be used giving the potential for a wider applicability range. Importantly, second- and third-generation CARs provide additionally costimulatory signals and enhance T cell activation, expansion, and in vivo persistence [4]. In principal, similarly to conventional CAR-T cells, T-iPSC can be also further genetically armed with cytokines, receptors, and other regulatory molecules in order to provide their derivatives with optimal immunotherapeutic properties such as enhanced proliferation and reduced exhaustion [2932]. Especially the rise of gene editing technologies, such as CRISPR/Cas9 and TALEN technologies, offers new perspectives for the multiplex modification of T-iPSC.

The potential of iPSCs to become a valuable source of readily available anti-tumor T cells depends on the development of a defined and efficient production process that could yield the cell numbers required for clinical application. In 2009, Timmermans et al. first reported the derivation of mature CD3+TCR+ T cells from human embryonic stem cells [25]. Since then, several groups have demonstrated successful in vitro generation of T lymphocytes from iPSC, although using slightly different methods [21, 23, 28]. However, all described protocols follow the same differentiation path, recapitulating the process of human T cell development. First, iPSCs are induced to form mesoderm from which definitive hemogenic endothelium arises as a next step. Hemogenic endothelium is then transitioning to form a pool of hematopoietic stem and progenitor cells (HSPC), a subpopulation of which has the potential to commit to the T cell lineage. The final important steps involve the emergence of CD8+/CD4+ DP cells and eventually of mature CD8 or CD4 SP T lymphocytes. Since this is a multi-step differentiation process where every developmental transition happens with different efficiency, one could imagine that the production of mature SP T cells for clinical applications is a challenge. In recent studies, investigators were able to generate enough numbers of anti-tumor T cells in order to test their functionality in vivo in xenograft murine models [26, 27, 28]. However, the development of an efficient method for the production of clinically relevant cell numbers is still not reported.

Another major challenge is that, although successful in generating T cells from iPSC or T-iPSC, most of the reported differentiation methods include the presence of uncharacterized serum and feeder cells of murine origin, which are not compatible with clinical level production. Induction of the hematopoietic program has been achieved through co-culture with OP9, a murine bone marrow stromal cell line, or C3H10T1/2, a mouse embryonic fibroblast line [21, 23, 33]. Further T lymphoid commitment requires the use of the same cell lines of murine origin transduced to overexpress the Notch ligands DLL1 or DLL4 [20, 21, 23, 33]. One could replace the murine feeder cells with cells of human origin, but until now attempts to create human-origin feeder cells for T cell development had disappointing results, as human fibroblasts or keratinocytes have failed to efficiently support the differentiation of human CD34+ to pro T cells or SP mature cells [34, 35]. Therefore, the development of a feeder-free and serum-free method is required. Kennedy et al. managed to replace feeder use, for the differentiation of iPSC towards CD34+CD43 HSPC, with a serum-free and stroma cell-free protocol based on embryoid body formation and the use of rationally selected cytokine combinations in a stepwise manner [20]. Creating an in vitro thymic niche by using plate-bound recombinant molecules of DLL4/DLL1 and VCAM-1, fused to the Fc portion of human IgG, has been used to generate T cell lineage cells from or cord blood-derived CD34+ cells [36], but the successful feeder-free differentiation of iPSC-derived HSPC has not been described.

Beyond antigen specificity, the functional potential of T cells depends also on their developmental maturity and their lineage subtype (, , -, CD4 or CD8, Treg, etc). This underscores once more the importance of developing differentiation protocols, which are based on the knowledge of human T lymphoid development, for the generation of the T cell subtype with the desired functionality. The first studies generating cytotoxic T cells from T-iPSC revealed that their phenotype and functionality was not precisely similar to that of mature CD8 T cells. The T-iPSCderived rejuvenated T cells although they were CD3+TCR+ showed T cellspecific gene expression profile and elicited specific cytotoxic responses against cells expressing the target antigen; they lacked expression of important surface molecules (such as CD2, CD5, CD28) and expressed high levels of innate T cell-related markers (such as CD56) [21, 23]. Also, Themeli et al. further reported that CAR-modified T-iPSC differentiate into CD3+TCR+CD8+ CAR-T cells whose gene expression profile was similar to that of -T cells [28]. Importantly, their in vivo anti-tumor functionality was analogous to that of peripheral blood-derived -T cells from the same donor bearing the same CAR [28]. Therefore, although the generated T-iPSCderived T cells express the endogenous TCR, they have phenotypic and functional characteristics of an innate-like lymphocyte. Similar lineage skewing has been observed in transgenic TCR mice [3739] and in vitro differentiation of TCR-engineered human CD34+ hematopoietic progenitors [40], wherein the emerging TCR+ T cells displayed innate T cell features, such as expression of CD8 and low levels of CD5 [37]. CD8 is expressed as a CD8 heterodimer on mature cytotoxic T cells while CD8 homodimers are present only on innate lymphocyte subtypes such as NK cells, -T cells, NK-T cells, or intestinal epithelial lymphocytes (IEL) [41, 42]. Heterodimeric CD8 has been shown to be a better coreceptor for TCR/pMHC binding than homodimeric CD8 [43] and the presence of CD8 is indicative of maturity. Indeed, T-iPSCderived T cells expressing CD8 exhibited improved antigen-specific cytotoxicity in vitro and in vivo compared with CD8 cells which show innate-like non-antigen-specific reactivity [27].

It has been suggested that premature expression of the transgenic TCR may prevent -selection of the cells, skew development towards the lineage, and result in the emergence of TCR-expressing T cells with properties [37, 38]. Interestingly, the pre-rearranged endogenous TCR of T-iPSC is already expressed on day 1520 of differentiation on OP9-DL1 [28], which is remarkably earlier than the appearance of TCR/ in differentiation of cord blood (CB)-CD34+ cells and some other reports on human ES/iPSC T cell differentiation [20, 25]. In addition, similarly to what is reported for the TCR transgenic mice, the generation of CD8-expressing DP cells from T-iPSC is very inefficient [21, 27, 33]. Interestingly, previous studies in transgenic murine models have demonstrated that lineage determination during T lymphoid differentiation is dependent on the synergy between TCR and Notch signaling and differences in Notch signal strength are also an important factor influencing versus development [44]. Murine T-iPSC differentiated in a 3D thymic culture generated antigen-specific anti-tumor T cells, which were phenotypically and functionally more similar to nave CD8 T cells in contrast to CD8 cells emerging from the OP9-DL1 co-culture [45]. Therefore, a thymic environment provides the correct combination of Notch and other signals that promote the maturation of thymocytes. Further research should focus on the development of in vitro differentiation systems that better mimic the interactions and the balanced TCR and Notch signaling that takes place within the thymus.

Phenotypic and functional evaluation of the T-iPSCderived CD8 T cells [27] or iPSC-derived CD8 T cells bearing a transgenic TCR [26] showed that they are similar to their peripheral blood counterparts. However, there are still gene expression discrepancies such as the lack of chemokine receptors (CCR7, CXCR3) and a weaker but still existing NK-like cytotoxicity [27]. In addition, it is still not clear whether their anti-tumor function is equivalent to that of conventional CD8 T cells. The in vivo anti-tumor functionality of regenerated CD8 T cells has been up to date tested in xenograft models where the tumor cells are inoculated intraperitoneally or subcutaneously and thus, not in a naturally occurring location limiting our insight on their migratory capacities [26, 27, 28, 33]. Finally, although treatment with iPSC-derived CD8 T cells significantly delayed tumor growth, it required multiple injections of high numbers of cells [26, 27, 33]. The use of xenograft models where tumor cells are inoculated at their natural sites and T cell-dose escalation could be more informative on the functionality of the iPSC-derived T cells.

NK cells constitute a robust part of the innate immune system implicated in recognition and lysis of malignant and virally infected cells. Their HLA-independent cytotoxic capacity makes them favorable candidates for off-the-shelf cellular therapeutic product compared with T cells. However, as already mentioned, their limited proliferative and genetic manipulation potential render their clinical application challenging. The perspective of manufacturing therapeutic NK cells from iPSC provides solutions to many of the bottlenecks of adoptive NK cell therapy.

Interestingly, the generation of NK cells from iPSC has proven easier and more straight-forward than the production of antigen-specific cytotoxic T cells. Although the first steps of differentiation towards HSPCs are similar to that of T differentiation, the commitment to NK lymphoid lineage is less complicated and does not require the presence of Notch signaling. Several studies have demonstrated the robust production of homogeneous, mature NK cells from human iPSC, which express all significant NK-defining markers such as CD56, FcRIIIa receptor (CD16), CD94, killer immunoglobulin-like receptors (KIRs), natural cytotoxicity receptors (NKp30, NKp44, and NKp46), activating receptors (NKG2D and DNAM-1), and death-inducing ligands (FasL, TRAIL) [46, 47, 48]. Most importantly, Knorr et al. reported a clinical-grade, serum-free, and feeder-free differentiation protocol to obtain functional NK cells from iPSC, which involves embryoid body formation in defined conditions and the use of membrane-bound interleukin 21-expressing artificial antigen-presenting cells [49]. According to the authors of the study, enough cytotoxic NK cells to treat a single patient could be produced from fewer than 250,000 input hiPSCs, thus facilitating the potential for clinical application in cancer therapy.

Apart from a mature phenotype, iPSC-derived NK cells display efficient cytotoxic capacity through direct receptor-mediated lysis, cytokine and chemokine secretion, and antibody-dependent cell-mediated cytotoxicity (ADCC) [46, 47]. When evaluating the anti-tumor activity in an ovarian cancer xenograft model, intraperitoneally injected iPSC-derived NK cells showed similar delay of tumor progression and overall survival as peripheral blood NK cells expanded on artificial antigen-presenting cells (aAPC) [47]. However, although statistically significant, this anti-tumor effect was not impressive and required multiple doses of NK cells. Interestingly, engineering iPSCs with a CAR bearing NK-specific costimulatory domains derived from NKG2D and 2B4 proteins optimized the targeted anti-tumor activity of the generated CARiPSC-NK cells and improved their in vivo expansion and cytotoxic capacity [48]. Importantly, a single dose of CARiPSC-NK cells resulted in less toxicity but similar anti-tumor effect as third-generation CAR-T cells against ovarian cancer in vivo, although comparison of tumor burden was limited to 3weeks post infusion in this study [48]. Further genetic engineering has been proposed in order to improve the function and therapeutic potential of iPSC-NK cells. For example, the expression of a non-cleavable CD16 would enhance ADCC potential, the addition of an IL15R-IL-15 fusion can provide self-stimulation, and expression of CXCR3 can improve homing of iPSC-NK cells [50].

Although generation of T and NK cells from iPSC overcomes many of the limitations of current manufacturing practices, their use would only really facilitate the applicability of adoptive cell therapy if they are available as a true universally applicable off-the-shelf product, which could be infused to any patient.

The major barrier limiting the applicability of allogeneic iPSC-derived products is the HLA-disparity between the effector T cells and the host, which may lead to graft rejection or graft-versus-host (GvH) reaction. Using matched previously banked iPSCs from HLA-homozygous donors as a starting material has been previously proposed [51]. It has been calculated that an iPSC bank with 50 HLA-homozygous iPSC lines could cover approximately 73% of the Japanese population [51] while 93% of the UK population would find a match within 150 HLA-homozygous lines [52]. However, the establishment of universal iPSC lines is considered to provide a true solution as they would provide widely applicable cellular products without the need for HLA-matching. Hypoimmunogenic, histocompatible pluripotent stem cell lines can be generated by elimination of HLA class I and II expression by disruption of 2m and CIITA gene respectively [53, 54, 55]. Allogeneic cells, which lack self class I HLA molecules, can however be rejected by host NK cells. Introduction of HLA-E, HLA-G, or of patient-specific HLA-C has been shown to reduce NK-mediated rejection of iPSC-derived cells [53, 54, 55]. Introduction of additional immunomodulatory molecules such as PD-L1 and CD47 can further reduce the recipients immune responses [55].

When using allogeneic T cells, the possibility of graft-versus-host reactions is a major concern. In order to avoid alloreactivity of iPSC-derived T cells, T-iPSC bearing an endogenous TCR of known specificity (virus- or cancer-specific) could be used. Alternatively, the surface expression of the TCR could be disrupted by the means of genome editing as previously described for conventional CAR-T cells [10, 56].

Although, many of the above genetic modifications have been already reported and evaluated in the iPSC level, there is up to date no study showing the generation of functional and mature lymphocytes from universal iPSCs.

The advancement of adoptive cell immunotherapy and the impressive clinical outcomes obtained targeting hematologic malignancies with CAR-T cells dictate for further developments towards a broader use of cellular therapeutics for more patients and more types of malignancy. The advent of iPSC technology provides new perspectives for the manufacturing of customized, tumor-targeting T/NK cells, with improved immunotherapeutic properties and the potential of universal off-the-shelf use (Fig. ). Rapid progress in the field of lymphoid differentiation of iPSC has brought the clinical application of iPSC-derived adoptive immunotherapy from theory to reality. Indeed, the first clinical trial testing an off-the-shelf, iPSC-derived NK cell product against advanced solid tumors started recruiting in 2019 (ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT03841110","term_id":"NCT03841110"}}NCT03841110). In addition, the production of iPSC-derived T cells and TCR/CAR-engineered T cells is already in pre-clinical development. Fate Therapeutics is developing TCR-less T-iPSCderived CD19-CAR-T cells where the CD19CAR is expressed from the TCR chain constant region (TRAC) locus, while Adaptimmune aims to develop off-the-shelf anti-tumor T cells from TCR-engineered iPSC.

However, there are still several challenges to be pre-clinically addressed before the first clinical application of iPSC-derived T cells. As mentioned above, the phenotypic and functional maturity of the generated T cell effectors has to be ensured as well as an anti-tumor potential comparable with natural T cells. Further, manufacturing protocols should be established which would allow for the efficient, GMP-grade, and clinical scale production of iPSC-derived T cell products. Finally, as with all iPSC-derived cellular products, the potential risk of malignant transformation due to contamination with undifferentiated iPSC has to be minimized, for example with the use of suicide genes such as the iC9/CID system [33]. Further future advances in iPSC and genome editing technologies in combination with in-depth knowledge of the fundamental mechanisms of T/NK cell function and the regulation of lymphoid development will provide the tools for the generation of iPSC-derived T/NK cell products with improved therapeutic anti-tumor function, better homing, persistence, and applicable across histocompatibility barriers.

Alexandros Nianias declares that he has no conflict of interest.

Maria Themeli reports serving as consultant for Covagen AG. In addition, Dr. Themeli has a patent WO2014165707A2 with royalties paid to Fate Therapeutics.

This article does not contain any studies with human or animal subjects performed by any of the authors.

This article is part of the Topical Collection on CART and Immunotherapy

Publishers Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Alexandros Nianias, Email: ln.cmumadretsma@sainain.a.

Maria Themeli, Phone: +31 (0) 204447413, Email: ln.cmumadretsma@ilemeht.m.

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Induced Pluripotent Stem Cell (iPSC)Derived Lymphocytes for Adoptive ...

Umoja Biopharma Presents Data on its Engineered Induced Pluripotent Stem Cell Platform at the 2022 International Society for Stem Cell Research Annual…

SEATTLE, June 16, 2022 (GLOBE NEWSWIRE) -- Umoja Biopharma, Inc., an immuno-oncology company pioneering off-the-shelf, integrated therapeutics that reprogram immune cells in vivo to treat patients with solid and hematologic malignancies, announced today that it will have a poster presentation at the 2022 International Society for Stem Cell Research (ISSCR) Annual Meeting, to be held June 15-18, 2022 in San Francisco, California.

On Wednesday, June 15th, Principal Scientist & iPSC Team Lead, Teisha Rowland, Ph.D., will give a poster presentation titled, A Synthetic Cytokine Receptor Platform for Producing Cytotoxic Innate Lymphocytes as Off-the-Shelf Cancer Therapeutics. The presentation will discuss Umojas engineered induced pluripotent stem cell (iPSC) platform, that incorporates the synthetic cytokine receptor system rapamycin-activated cytokine receptor (RACR) platform. Umojas engineered iPSCs that are modified to express RACR, called RACR-induced cytotoxic innate lymphoid (iCIL) cells, drive differentiation and expansion of the cells while eliminating the need for expensive cytokines and other raw materials. The RACR platform has the potential to enable cytokine-free manufacturing and engraftment of the engineered cells in the patient without the need for toxic lymphodepletion.

Despite the advances chimeric antigen receptor T cell therapies have provided to the oncology space, we continue to battle significant challenges that these therapies cannot address, like limited expansion capacity and scalability, manufacturing complexity, variability among patients, and the need for toxic chemotherapy administration to combat patients anti-allograft response, said Andy Scharenberg, M.D., co-founder and Chief Executive Officer of Umoja. We are developing an engineered iPSC platform, including the RACR platform, to address these challenges by enabling a scalable, virtually unlimited, and simplified manufacturing of engineered, cancer-fighting cytotoxic innate lymphocytes.

About Umoja Biopharma

Umoja Biopharma, Inc. is an early clinical-stage company advancing an entirely new approach to immunotherapy. Umoja Biopharma, Inc. is a transformative multi-platform immuno-oncology company founded with the goal of creating curative treatments for solid and hematological malignancies by reprogramming immune cells in vivo to target and fight cancer. Founded based on pioneering work performed at Seattle Childrens Research Institute and Purdue University, Umojas novel approach is powered by integrated cellular immunotherapy technologies including the VivoVec in vivo delivery platform, the RACR/CAR in vivo cell expansion/control platform, and the TumorTag targeting platform. Designed from the ground up to work together, these platforms are being developed to create and harness a powerful immune response in the body to directly, safely, and controllably attack cancer. Umoja believes that its approach can provide broader access to the most advanced immunotherapies and enable more patients to live better, fuller lives. To learn more, visit http://umoja-biopharma.com/.

About RACR

CAR T cells generated by the body with VivoVec can be expanded and sustained with the rapamycin activated cytokine receptor (RACR) system, an engineered signaling system designed to improve chimeric antigen receptor (CAR) T cell persistence and produce durable anti-tumor responses. The RACR/CAR payload is integrated into the genomic DNA of a patients T cells. Rapamycin activates the RACR system resulting in preferential expansion and survival of cancer-fighting T cells. The RACR technology enables a patients cells to expand in a manner that resembles a natural immune response that does not require lymphodepletion, promoting durable T cell engraftment. RACR/CAR technology can also be used to enhanceex vivomanufacturing in support of more traditional autologous or allogeneic cell therapy manufacturing processes. To learn more about Umojas RACR platform please visit https://www.umoja-biopharma.com/platforms/

Media Contact: Darren Opland, Ph.D. LifeSci Communications darren@lifescicomms.com

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Umoja Biopharma Presents Data on its Engineered Induced Pluripotent Stem Cell Platform at the 2022 International Society for Stem Cell Research Annual...

UB-led study presents critical step forward in understanding Parkinson’s disease and how to treat it – UBNow: News and views for UB faculty and staff…

A new study led by a researcher in the Jacobs School of Medicine and Biomedical Sciences at UB has important implications for developing future treatments for Parkinsons disease (PD), a progressive nervous system disorder that affects movement and often includes tremors.

In this study, we find a method to differentiate human-induced pluripotent stem cells (iPSCs) to A9 dopamine neurons (A9 DA), which are lost in Parkinsons disease, says Jian Feng, professor of physiology and biophysics in the Jacobs School and senior author on the paper published May 24 in Molecular Psychiatry.

These neurons are pacemakers that continuously fire action potentials regardless of excitatory inputs from other neurons, he adds. Their pace-making property is very important to their function and underlies their vulnerability in Parkinsons disease.

This exciting breakthrough is a critical step forward in efforts to better understand Parkinsons disease and how to treat it, says Allison Brashear, vice president for health sciences and dean of the Jacobs School. Jian Feng and his team are to be commended for their innovation and resolve.

Feng explains there are many different types of dopamine neurons in the human brain, and each type is responsible for different brain functions.

Nigral dopamine neurons, also known as the A9 DA neurons, are responsible for controlling voluntary movements. The loss of these neurons causes the movement symptoms of Parkinsons disease, he says.

Scientists have been trying hard to generate these neurons from human pluripotent stem cells to study Parkinsons disease and develop better therapies, Feng says. We have succeeded in making A9 dopamine neurons from human induced pluripotent stem cells. It means that we can now generate these neurons from any PD patients to study their disease.

Feng notes that A9 DA neurons are probably the largest cells in the human body. Their volume is about four times the volume of a mature human egg.

Over 99% of the volume is contributed by their extremely extensive axon branches. The total length of axon branches of a single A9 DA neuron is about 4.5 meters, he says. The cell is like the water supply system in a city, with a relatively small plant and hundreds of miles of water pipes going to each building.

In addition to their unique morphology, the A9 DA neurons are pacemakers they fire action potentials continuously, regardless of synaptic input.

They depend on Ca2+ channels to maintain the pace-making activities. Thus, the cells need to deal with a lot of stress from handling Ca2+ and dopamine, Feng says. These unique features of A9 DA neurons make them vulnerable. Lots of efforts are being directed at understanding these vulnerabilities, with the hope of finding a way to arrest or prevent their loss in Parkinsons disease.

Pace-making is an important feature and vulnerability of A9 DA neurons. Now that we can generate A9 DA pacemakers from any patient, it is possible to use these neurons to screen for compounds that may protect their loss in PD, he notes. It is also possible to test whether these cells are a better candidate for transplantation therapy of PD.

To differentiate human iPSCs to A9 DA neurons, the researchers tried to mimic what happens in embryonic development, in which the cells secrete proteins called morphogens to signal to each other their correct position and destiny in the embryo.

Feng notes the A9 DA neurons are in the ventral part of the midbrain in development.

Thus, we differentiate the human iPSCs in three stages, each with different chemicals to mimic the developmental process, he says. The challenge is to identify the correct concentration, duration and treatment window of each chemical.

The combination of this painstaking work, which is based on previous work by many others in the field, makes it possible for us to generate A9 DA neurons, he adds.

Feng points out there are a number of roadblocks to studying Parkinsons disease, but that significant progress is being made.

There is no objective diagnostic test of Parkinsons disease, and when PD is diagnosed by clinical symptoms, it is already too late. The loss of nigral DA neurons has already been going on for at least a decade, he says.

There was previously no way to make human dopamine neurons from a PD patient so we could study these neurons to find out what goes wrong.

Scientists have been using animal models and human cell lines to study Parkinsons disease, but these systems are inadequate in their ability to reflect the situation in human nigral DA neurons, Feng says.

Just within the past 15 years, PD research has been transformed by the ability to make patient-specific dopamine neurons that are increasingly similar to their counterparts in the brain of a PD patient.

Houbo Jiang, research scientist in the Department of Physiology and Biophysics, and Hong Li, a former postdoctoral associate in the Department of Physiology and Biophysics, are co first-authors on the paper.

Other co-authors are Hanqin Li, a graduate of the doctoral program in neuroscience and currently a postdoctoral fellow at University of California, Berkeley; Li Li, a trainee in UBs doctoral program in neuroscience; and Zhen Yan, SUNY Distinguished Professor of Physiology and Biophysics.

The study was funded by the Department of Veterans Affairs, National Institutes of Health and by New York State Stem Cell Science (NYSTEM).

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UB-led study presents critical step forward in understanding Parkinson's disease and how to treat it - UBNow: News and views for UB faculty and staff...