A Review on Leukemia and iPS Technology: Application in Novel Treatment and Future

Amirhosein Maali1, Amir Atashi2, Sasan Ghaffari3, Reza Kouchaki1, Fereshteh Abdolmaleki1, Mehdi Azad1*

1Faculty of Allied Medicine, Qazvin University of Medical Sciences, Qazvin, Iran2Stem Cells and Tissue Engineering Research Center, Shahroud University of Medical Sciences, Shahroud, Iran3Department of Hematology, Faculty of Allied Medicine, Tehran University of Medical Sciences, Tehran, Iran

*Corresponding Author: Mehdi Azad. PhD. Qazvin, Shahid Bahonar Boulevard, University of Medical Sciences, Faculty of Allied Medicine (Hematology), Department of Medical laboratory sciences. Tel: +982833359501 Fax: +982833338034 Zip code: 3419759811Email Address: haematologicca@gmail.com

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Abstract

Leukemia is an uncontrollable growth of hematopoietic cells due to a mutation in DNA followed by cellular dysregulation and one or more chromosomal disorder that generally leads to a clonal abnormality. Theoretical and technical inability in early screening and distinguishing cancer, tumor tolerance to common treatment methods, repeated relapses of cancer after remission phase, heterogeneous chromosomal abnormality, and the side effects of current chemotherapies are some challenges that we face with leukemia and other malignancies. Induced pluripotent stem cells (iPSC) opened a promising window to a wide range of diseases, including leukemia. Overcoming the barriers in leukemia is possible with iPSC technology. Induced hematopoietic stem cell transplantation (and gene therapy), induced cytotoxic T-lymphocytes and reprogrammed NK cells that strengthen the immune system, miRNAs, modeling approaches, and supportive cares are aspects of the novel treatment based on iPSC technology.

Keywords: Leukemia, iPS, treatment, Reprogrammed NK cells, miRNAs, gene therapy, iHSC, malignancy modeling

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1. Introduction

Cancer is the uncontrollable cell growth, which is one of the leading causes of death worldwide. According to published statistics, cancer is the second and third reason for death in developed and developing countries, respectively.

Although in recent decades, significant developments in cancer diagnosis and treatment have been achieved, many areas in this field are still uncharted. Rudimentary knowledge and basic methods of cancer screening, imperfect treatment processes, and tumors tolerant to common treatments are all challenges that we face with a malignancy. Furthermore, repeated post-remission relapses, heterogeneous chromosomal abnormality, and malignant cells’ plasticity only serve to exacerbate the situation [1-3].

Cancer, almost, originates from one or more chromosomal disorder, which will generally lead to a clonal abnormality, a change in the gene expression profile, cell-cycle dysregulation, apoptosis, and ultimately incoherent cell growth. The genetic disorders that occur in intracellular key molecules and proto-oncogene and tumor suppressor genes result in malignancy. Since roughly 2004, the researchers’ efforts toward better understanding of cancer biology have evolved and improved the approach to prognosis, diagnosis, and treatment of cancer [4]. Naturally, chromosomal disorders arise before tumor forms in the body, and if they could be diagnosed before the tumor took place, it would be much easier to deal with and control cancer [5].

2. Review to Leukemia and Its Variants

Leukemia also occurs due to a mutation in DNA followed by interference in cellular function. The exact reason for each leukemia has not been recognized yet, but it is believed that its inception has environmental and inheritable reasons. There are four major classes of leukemia:

2.1. Acute Lymphoblastic Leukemia (ALL) is a relatively rare type of leukemia that leads to an increased number of WBCs and lymphoid cells in the peripheral blood and bone marrow (BM). Common translocations in ALL are Philadelphia (in 1.6% of patients; weak prognosis), t(4:11)(q21:q23) (in 1.6% of patients; weak prognosis), t(8:14)(q24:q32) (rare prevalence; weak prognosis), t(1:9)(q23:p13) (in 4.8% of patient), t(12:21) (in 25.4% of patients), t(11:14) (p13:q11) (rare prevalence). Other aberrancies include low-level hypodiploidy (weak prognosis), high-level hyperdiploidy (especially trisomy 4, 10, 17 with favorable prognosis), del(9p) (favorable prognosis), and complex karyotype (weak prognosis) [6-14].

Diagnosis of the affected lymphoid cell line plays a key role in choosing treatment protocol. Utilizing immunophenotyping assays can help to distinguish the type of ALL (prevalence of B-ALL and T-ALL is %80 and %20, respectively) [15, 16]. The B-ALL immunophenotypic profile is determined by specific B-cell CD markers, such as CD19, CD20, CD22, and CD79a. Primary B-cell blasts are generally CD10+ and CD34+, and more mature neoplastic cells express high levels of CD20. This can particularly be used to estimate the commencement of the disease. To identify the immunophenotypic profile of T-ALL, CD7, CD3, CD5, CD2, and CD1a can be used [17]. ALL in children has high incidence rate and the cure-rate (CR) is achieved in 75 to 90% of the patients by unique treatment methods [18-20].

2.2. Chronic lymphoblastic leukemia (CLL) is a type of leukemia that is accompanied by clonal proliferation and amassing of mature lymphocytes in peripheral blood, bone marrow, and lymph nodes [21]. Leukemic transformations in CLL are coupled with genetic changes and deletions of specific miRNA genes, developing survival properties of T and B-cell and resistance to apoptosis [22-24].

Long arm deletions of chromosome 13, especially 13q14 band (del (13q14)), are seen in more than 55% of cases. Recently, it has been shown that miR16-1 and miR15a are in the deleted areas (13q14) [22]. Other common aberrancies include long arm deletion of chromosome 11 (del (11q)) (in 25% of patients) [25, 26], trisomy 12 (in 10 to 20% of patients) [27], and short arm deletion of chromosome 17 (del (17p)) (in 5% of patients) [28].

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Primary diagnosis of CLL is accomplished by morphologically inspecting lymphocytes and witnessing a count of 5000 cells/µl of peripheral blood for at least 3 consecutive months. Most of the leukemic lymphocytes that can be seen in peripheral blood smear are tiny, mature cells with small cytoplasm and condensed nucleus. These cells may be present in combination with large and atypical cells, or with prolymphocytosis (ultimately up to 55% of the peripheral blood lymphoid cells). Lymph node agitation, lack of immunity, anemia, and coagulation disorders are the features of CLL [29]. In today’s society, this leukemia is widespread and is more prevalent in people over the age of 55. The 5-year survival rate in these individuals with advanced treatments is up to 75% [30].

2.3. Acute Myeloblastic Leukemia (AML) is followed by a heterogeneous disorder with clonal expansion of myeloid progenitor blasts in bone marrow and peripheral blood. The molecular screening has the main role in diagnosis, prognosis, and treatment process in AML. Genetic abnormality is seen in 50% of AML patients [31]. For example, the 5, 7, and 11q23 chromosomal changes and complex karyotype (simultaneous three-chromosomal abnormalities) are accompanied by lower response to treatment and bad prognosis, whereas abnormalities like t(15:17)(q22:q12), t(8:21)(q22:q22), and inv(16)(p13.1:q22) have more favorable prognosis [32, 33]. However, about 40 to 50% of AML patients are cytogenetically normal, named CN-AML (cytogenetically normal AML) [32]. Recently, utilizing gene sequencing to investigate mutations has been a powerful tool to analyze AML and treat AML patients. This disorder is reported more in children and men, respectively, than in adults and women [34]. Previously, AML had a very poor prognosis, but with the aid of improved treatment regimens and supportive cares, such as antibiotics and blood transfusion, AML has reached 35 to 40% CR for people under the age of 60 [33]. Indeed, the prognosis has been better in people over 60 years, but it is not sufficient.

2.4. Chronic Myeloblastic leukemia (CML) is a type of myeloproliferative disorder accompanied by a massive invasion of peripheral blood, bone marrow, and spleen by mature and immature myeloid cells. Currently, in more than 90% of cases, CML, which is known for a prominent increase in normal myeloid cells in both activity and function, is diagnosed in preliminary chronic phase (CML-CP). If CML is not treated, it will enter the accelerated phase and then the more aggressive blastic phase (BP). The most common genetic translocation in CML is reciprocal t(9:22)(q34:q11). Consequent recombinant protein, BCR-ABL1, has persistent tyrosine kinase activity that begets malignancy [35]. In-vitro studies have shown that BCR-ABL1 is an oncogene that causes the growth of malignant cells and suppression of apoptosis. Considering its importance, many CML treatments focus on this protein. This leukemia is seen more in adults, and up to 90% CR is reported [36].

3. iPS Technology: a Grand Medical Evolution

Stem cells are identified by two characteristics: the ability of self-renewal and plasticity (the differentiation potential) [37]. These abilities are the most important factors in cell therapy. Different plasticity potential of stem cells divides them into 5 groups, including totipotent, pluripotent, multipotent, oligopotent, and unipotent [38, 39].

Researchers deduced that pluripotency in pluripotent stem cells is influenced by a complex network of regulatory genes. In 2006, Takahashi and Yamanaka reached a new technology that allows the integration of transcription factors responsible for pluripotency making it possible to produce pluripotent embryonic cells by editing somatic cells’ genes. This process, that is the opposite of differentiation, is called reprogramming and the produced cells are named induced pluripotent stem cell (iPSC). In this project, human and mouse somatic cells return to embryonic state after receiving and subsequently expressing OCT4, KLF4, SOX2, and C-MYC (OKSM) transcription factors (Table 1) [40-42].

OCT

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5

Table 1. Genes relevant to iPSC production and their chromosomal location. OKSM factors, NANOG, and LIN-28 factors have a part in pluripotency status. HOXA9, HOXA5, RORA, SOX4, ERG, GATA2, GFI1b, FOS, ETV8, and MYB are important in iHSC potency and GATA3, RUNX3, TBX21, and IKZF1 are involved in induced immunologic cells.

The constructed iPSC can differentiate into whole somatic cells. Transplanting differentiated cells can eliminate disease-causing cellular defects. These cells have also been utilized in disease modeling and drug discovery. The upside is there is no immunological rejection risk (autologous graft), no limitation in the number of required cells, and no ethical issues. The first vector used in cloning the OKSM factors was retroviral vector [43, 44]. There are many challenges in preclinical trials of iPSC, one being teratoma development. Integrating vectors can promote tumorigenesis if they are inserted in sensitive sites like promoters and enhancers of host gene [45]. One solution to overcome this challenge is non-integrating vectors and molecules [46-60]. Since making iPSC is a long-term process, in urgent situations, it is possible to use iPSC of a fully MHC-matched donor from stem cell banks, which are expanding every day [61].

In 2007, the first clinical trial in the field of iPSC-based therapy was conducted in treating sickle cell anemic mice by autologous transplantation [62]. Nevertheless, it is clear that there is a long way to reach human iPSC-based therapy, but many kinds of research in this field are being undertaken, and so we will discuss these studies in the following sections [47].

3.1. Hematopoietic stem cell transplantation and gene therapy based on iPSCs

Hematopoietic stem cell (HSC) or hematoblast is a multipotent stem cell that can differentiate to all blood cell lines through hematopoiesis [63]. Hematopoietic stem cell transplantation (HSCT) is the most advanced and effective method of leukemia treatment [64, 65]. HSCT can be used to treat malignant disorders, such as ALL, AML, CML, Hodgkin lymphoma (relapsed/refractory), Non-Hodgkin lymphoma, myelodysplastic syndrome, multiple myeloma (MM); and also nonmalignant disorders such as thalassemia, sickle cell anemia, Fanconi anemia, aplastic anemia, immunodeficiency syndromes, and congenital metabolic disorders. Nowadays, HSC transplantation is practiced in both allogeneic and autologous donations [66].

As mentioned before, the most important clinical challenge in HSCT is the lack of compatible donors in the golden time of transplant and also immunological rejection risk [67]. Subcutaneous injection of granulocyte colony stimulating factors (G-CSF), such as Filgrastim and Lenograstim, which are suspected to increase the risk of neoplasia, discharge HSC from BM to peripheral blood [68]. After Yamanaka studies in the field of reprogramming, which led to the invention iPS technology, many researchers have been trying to produce induced hematopoietic stem cells (iHSC) derived from iPSC [69]. In fact, iPS technology advancements have made the creation of healthy pluripotent (Subsequently multipotent) stem cells from patients with blood malignancy possible [70]. To produce these cells, Szabo et al. showed that the sole expression of OCT4 of OKSM factors can generate HSC from fibroblasts [69]. Doulatov et al. also reprogrammed myeloid cell line to HSC in a similar project using 13 transcription factors [70]. In the treatment of congenital-based leukemia, genetic disorders must be rectified. To this end, new genetic engineering tools can be used, including CRISPR-Cas9, zinc-finger nucleases (ZFN), meganucleases, transcription activator-like effector nucleases (TALEN) and double-strand break (DSB) nucleases [71-74]. In gene regulatory network complex, two transcription factors (HOXA and ERG) act as the main differentiation and proliferation factors in HSC. These two factors are inducers of the self-renewal ability and are thus oncogenic. Certainly, dysregulation of these factors develops a wide range of leukemic cells. Studies show that MYB, SOX4, RORA, HOXA5, and HOXA9 are all part of gene regulatory complex [70].

The classic marker of human iHSC is CD34 [75]. Many cluster of differentiation markers such as CD90, CD38, CD34, CD45, CD133, CD105, and C-kit (stem cell receptor) help distinguish HSC [76]. To ensure credible purification, cytochemistry and flow cytometry assays can be used, and the FACS technique is also useful to purify iPSCs and iHSCs. The safe, effective, and highly pure product ensures an HSCT with the least possibility of side effect.

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3.2. Induced cytotoxic T-lymphocytes

During recent years, cancer immunotherapy has expanded by utilizing various technologies generally based on reversing immunosuppression [77-79], transferring cytotoxic T lymphocyte (CTL) [80-82], transferring TCR gene [83-85], and so on. Before producing iPSC-derived CTL (iCTL), it is possible to differentiate them into double positive cells (DP: CD4/8+) in op9 culture media [86, 87]. The major cytokine in T-cell differentiation is IL-2 [88, 89]. After DP purification, cells can be stimulated and differentiated to CD8αβ+. By analyzing RNA-seq, researchers realized that iCTL also expresses other surface markers including TBX21, RUNX3, GATA3, and IKZF1 in levels as high as CD8+ [87].

Due to the unlimited in-vitro expansion of iCTL, it is possible to provide iCTL based on the need of each therapeutic panel, and iPS technology has made it feasible to prepare autologous CTL from patients’ own somatic cells. When T-cell is reprogrammed from iPSC, the TCR gene, which responds to a specific antigen, is propagated inside the cell. Therefore, all iCTLs express the same TCR on their surface [90]. The iCTL has cytotoxic capabilities against particular proteins in leukemia, like Wilms’ Tumor protein1 (WT1) (in AML-HL60 and THP1 cell line). Certainly, cells that do not express MHC-I on their surface escape CTL cytotoxic effects even if WT1 is expressed. For the first time, iCTL was used to treat NOG mice with AML-HL60 cell line. The cured mice clearly showed longer lifespan compared with the control group. In the treated mice, iCTL was not oncogenic nor was it tumorigenic. Also, it did not initiate any tissue damage [87].

The advent of iPS technology and then the invention of iCTL have been an accomplishment to help us with new methods in the cellular immunotherapy of cancer. In addition to using iCTL against malignant cells in cancer and leukemia, it is also used in resistant infections. Considering high specificity of iCTL, lower risk of immunologic rejection (due to being autologous), and high efficiency and purity, it could develop a great shift in the treatment of cancer. iCTLs can be used as an agent to prevent cancer relapse. So, iCTL application is promising in treating and monitoring leukemic patients.

3.3. Reprogrammed NK cells

Natural killer cells (NK cells) are a part of immune system that secrete cytokines and enzymes to perforate and lyse invading cells. NK cells are the link between adaptive and innate immune systems that can enhance and accelerate immunologic responses using their memory ability [91].

Cell therapy has helped to improve the natural immune system by using ex-vivo cultured NK cells. Currently, these cells are extracted from the blood circulation by leukapheresis and then separated from other cells via magnetic beads coated with anti-CD56. After purification, it is possible to expand them in culture media with B cell line transformed with EBV, or K562 cell lines that express co-stimulatory molecules and IL-21 and IL-5 [92]. However, utilizing this ex-vivo growth method cannot produce stable NK cells in-vivo to confront malignant cells, so achieving a durable and efficient NK cell in-vitro is sorely needed [91]. According to the studies on induced NK cells based on iPSC (iNK cells), it is possible to acquire stable and effective NK cell in-vitro using IL-7 and FLT3L for differentiation [44, 49, 93]. For the first time, the use of iNK cells in-vivo has been successful in OVA mice (as a tumor of artificial Ag) and NOG mice (with AML-OVN1 and MEGO1 cell line) [91]. As in iCTL, reprogrammed NK cell can supply the desired volume of cell product to be used in leukemia therapy panel without any immunological response.

3.4. miRNA and iPSC

The microRNAs (miRNA) are a group of small interference RNA (19 to 22 nucleotide) that innately control protein-coding genes. Studies regarding this RNA has proven their role in the onset of tumorigenesis and tumor metastasis. Considering their role as oncogene and tumor suppressor, there are many possible therapeutic strategies to cure malignancies based on miRNA [94, 95].

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204205206207208209210

211212213214215216217218219

220221222223224225

226

227

228229230

231232233234235236237238239240241

242

243244245246

Induction of miRNAs correlated with oncogenic genes and inhibition of miRNAs related to tumor suppressor RNAs bursts progenitor cells count and starts malignancy. While miRNA is critical in intracellular processing, recent studies have emphasized its extracellular effects. These miRNAs are produced and secreted like hormones from cells in the microenvironment and are eventually released to blood circulation [96]. Secreted miRNA is taken up by cells in various parts of the body as they regulate protein expression prior to translation [97].

The iPS technology delivers new methods in miRNA-based therapies. As repeatedly said, iPS cells are pluripotent with self-renewal and differentiation potential. So, being able to derive them from hematopoietic cells and their ability to secrete particular miRNAs could pave the road in the future for target therapy of leukemia and other cancers [98]. Furthermore, miRNA has a direct role in regulating immune responses. For instance, transferring miR335 from T-cell to synaptic space created through Ag presentation has been done to neutralize intracellular reaction of target cell [99]. The iCTL (explained previously) with high expression potential of miR335, could be a part of compound therapy against malignant cells based on target therapy, immunotherapy, and cell therapy. In addition to the measures above, using miRNA has made it possible to combine iPS technology with current strategies in dealing with cancers, including sandwich RNAi inhibition, micro mimic agent complex RNAi inhibition, small molecules inhibitors of miRNAs (SMIRs), and targeting miRNA from microvesicle and exosome [94, 100].

4. Hematopoietic Malignancy Modeling

As mentioned before, one aspect of iPS technology is identifying the molecular pathway of different diseases to better understand their pathogenesis, and present distinctive therapy for them. Patients’ iPS cells, which differentiate to a specific cellular line of the current disease, can be used for in-vitro modeling, drug synthesis, appropriate dosage determination, and toxicity evaluation [101-103]. Separating the mutant cells from the bloodstream of the patient and reprogramming them to iPSC makes hematopoietic malignancy modeling possible. Being able to identify the translocation patterns in leukemic cells using only one cell derived from cancer progenitor cells is accomplished because of the stability of genetic content through reprogramming.

Currently, with access to these cells, assessing the epigenetics of malignancies and target therapy is within reach. For example, oncogenic translocations (including silencing of tumor suppressor genes, changing proto-oncogene to oncogene or dysregulation of apoptotic genes) and epigenetic dysregulations (changing DNA methylation pattern and modification of miRNA expression) can change the expression of progenitor cell genome leading to malignancy development. It has been shown that blocking the Hedgehog signaling could stop the growth of tumor in MM, CML, pancreas cancer, and brain tumors [104, 105]. Also, it has been demonstrated that NOTCH and Wnt signaling are involved in T-ALL, CML, and medulloblastoma [106]. In another example, human cancers generally show an increment of telomerase activity; this cellular process indicates that limiting telomerase activity likely prompts apoptosis and reduces self-renewal activity of mutant cells in MM [107].

So far, extensive leukemia modeling activities have been carried out using iPS technology including:

4.1. iPSC in AML: In 2017, Zhu et al. made the iPSC line derived from AML-M6 cells in-vitro and identified their biological properties and illustrated that these iPSCs are pluripotent and can differentiate into the three germ layers [108]. Also in 2015, Salci et al. produced HSC from fibroblasts of AML patients using iPS technology and compared their cytogenetic, immunophenotype, function, and morphology with their myeloid blasts. They showed that these HSCs have normal progenitor status and do not have leukemic mutations [109]. But In 2017, while Chao et al. were generating iPSC from MLL-rearranged AML patients, cells retained their cytogenetic and mutagenic abnormalities, but their epigenetic status had reversed back to normal. Therefore, there is no leukemic potential, but they regain the methylation patterns and leukemic properties upon re-differentiation to hematopoietic cells [110, 111]. In another study in 2014, Liu et al. succeed in making murine AML models. They created mice with chimeric iPSCs by overexpressing human MLL-AF9 fusion gene in hematopoietic cells containing OKSM factors. These mice spontaneously developed AML because of MLL-AF9 reactivation. This experiment showed that epigenetic

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247248249250251

252253254255256257258259260261262

263

264

265266267268269270271

272273274275276277278279280

281

282283284285286287288289290291292

regulators that manipulate the MLL-AF9 gene expression pattern are the bridge between MLL-AF9 leukemia cells and derived iPSCs [112].

4.2. iPSC in FPD/AML: In 2014, Sakurai et al. derived iPSCs from FPD/AML patients. They showed that in this situation hematopoietic progenitor cells’ formation and megakaryocyte differentiation are incomplete. For the first time, they showed the role of RUNX1 in human hematopoiesis and the effect of heterozygous mutant RUNX1 in megakaryocyte differentiation. They also showed that FPD/iPSC phenotypes are the result of the haploinsufficiency of RUNX1 [113]. In 2015, Lizuka et al. showed that RUNX1 is associated with thrombocytopenia in FPD/AML patients. Because HSC-derived FPD/iPSC had a dysregulation in megakaryocyte differentiation, and they corrected megakaryopoiesis by editing the RUNX1 mutation via TALEN [114].

4.3. iPSC in CML: In 2012, Kumano et al. produced iPSCs from imatinib-sensitive CML patients. These CML IPSCs continued to express the BCR-ABL onco-protein but were resistant to imatinib. They were shown to maintain iPSC signaling that compensate for the inhibition of BCR-ABL. Of course, these cells regained susceptibility to imatinib after being converted to the hematopoietic lineage [115].

4.4. iPSC in ALL: In 2016, Munoz-Lopez et al. attempted to produce iPSCs from the B-ALL subtypes, but neither the primary blasts nor the B-ALL cell lines (even with adding boosters) did not have reprogramming potential to achieve the pluripotency status. However, they were able to reprogram MLL-AF4-overexpressing HSCs/B-progenitors. This suggests that cells origin and leukemic genes were not the issue [116]. In 2016, Zhang et al. showed that the apoptosis, NF-κB, DOT1L, and LSD1 pathways were richer in reprogramming-incompetent colonies than reprogramming-competent colonies. The inhibitors of these pathways increased reprogramming efficiency in T-ALL cells. This indicates that these pathways are barriers to reprogramming in T-ALL cells [117].

4.5. iPSC in NS/JMML: In 2015, Mulero-Navarro et al. found that the myeloid cells derived from NS/JMML human induced pluripotent stem cells (hiPSC) had increased STAT5 signaling and two up-regulated microRNAs (miR-223 and miR-15a) that distinguish JMML caused by PTPN11 mutation from other genetic forms of this disease. Reducing the miR-223 activity causes normalization of the myelogenesis in the NS/JMML hiPSCs. Also, microRNA target gene expression levels were reduced in hiPSC-derived myeloid cells as well as in JMML cells with PTPN11 mutations (Table 2) [118].

Disease Year Type Origin Reprogramming factors

Vector Investigate Scientists References

CML 2012 Modeling - OKSM Retroviral CML modeling and study on pathophysiologic effects

Kumano K. et al. [115]

FPD/AML 2014 Modeling Peripheral T-cell

OKSM Sendaiviral Study on physiologic roles of RUNX1 in human hematopoiesis and FPD/AML pathophysiology

Sakurai M. et al. [113]

AML 2014 Modeling LIN- bone marrow cells

OKSM Retroviral Generation of AML-murine with overexpression of human MLL-AF9 fusion gene

Lui Y. et al. [112]

AML 2015 Modeling FibroblastOKSM

Lentiviral AML modeling and generating the healthy hematopoietic progenitors

Salci K.R. et al. [109]

NS/JMML 2015 Modeling Fibroblast OKSM Retroviral JMML modeling by human iPSC derived from NS/JMML and non-leukemic patients with PTPN11 mutation

Mulero-Navarro S. et al.

[118]

FPD/AML 2015 Modeling (gene editing)

Fibroblast OKSM Retroviral iPSC generation from a FPD/AML patient and modeling (with and without gene correction) via TALEN

Iizuka H et al. [114]

T-ALL 2016 Modeling LIN- bone marrow cells

OKSM Retroviral Identification of reprogramming blockage pathways in T-ALL

Zhang H. et al. [117]

9

293294

295296297298299300301

302303304305

306307308309310311312

313314315316317318

B-ALL 2016 Modeling Myeloid cells

Polycistronics:1)lentiviral OKSM2)sendai OKSM

Monocystronics:1)Episomal OKSM or OKSL2)retroviral OKSM3)sendai OKSM

iPSC generation from B-ALL subtypes Munoz-Lopez A. et al.

[116]

AML(M6) 2017 Modeling Infiltrated skin

OKSM Lentiviral iPSC generation from AML cell line Zhu L.F. et al. [108]

AML 2017 Modeling-

OKSM Sendaiviral AML-iPSC generation and study on its epigenetic and genetic properties

Chao M.P. et al. [110]

Table 2. Recent iPS projects in the field of modeling of leukemia. NS: noonan syndrome; JMML: juvenile myelomonocytic leukemia; FPD/AML: familial platelet disorder with propensity to acute myeloid leukemia; LIN-: Lineage negative; OKSM: OCT3/4, SOX2, KLF4, c-MYC; OKSL: OCT3/4, SOX2, KLF4, LIN28.

Thus, understanding the pathogenesis and complete etiology of leukemia can help design more specialized drugs against malignant cells with lower side effects. For example, the drugs interfering with cell signaling will stimulate tumor suppressor genes or repress particular oncogenes. However, the systemic cytotoxicity of cell signal-interfering drugs has always been a major concern. It means that drug therapy influences both cancer cells and normal hematopoietic stem cells [107, 119, 120]. Providing malignant leukemia cells in a non-invasive way for in-vitro studies with great efficiency and in desirable abundance cannot be done so easily. This problem could be overcome by the application of iPS technology and reprogramming. Using malignant hematopoietic stem cells derived from iPSC will serve as a great tool in fighting leukemia in the fields of drug screening and monitoring, new drug designing, dosage determination for patients, and molecular studies.

5. Supportive Care by iPSC-derived Megakaryocytic and Erythropoietic Stem Cells

In most cases of leukemia, patients suffer from comorbidities resulting from the myelophthisic status. Occupying the existing niches in BM scaffolds by mutant and malignant cells diminishes the space needed for growth of normal cell types, such as erythroid cells and platelets. In addition, in some leukemias, such as AML-M6 and AML-M7, the direct destruction of erythroid and platelet lineage, respectively, are observed. Thus, in leukemic patients, symptoms related to erythropenia and thrombocytopenia, such as gastrointestinal hemorrhages, petechiae, purpura (and other coagulation disorders), respiratory distress (due to decreased RBC), lethargy, severe anemia, and drowsiness are frequently observed [122]. Sometimes, these symptoms are the main reason for patient referral to a physician, which is followed by leukemia diagnosis.

This problem could be overcome, in the future, by applying iPS technology. In 2012, Kobari et al. illustrated complete in-vitro terminal maturation of erythroid lineage from hiPSCs and subsequent normal production of Hb in-vivo by injection of hiPSCs into mice. This finding indicates that iPSC can be a supplemental source of RBCs. Their other goal was to produce modified functional RBCs from the sickle-cell IPSC without any gene editing and drug therapy [123]. In 2011, Dias et al. showed that the production of RBC from transgene-free human iPSC has similar efficiency compared with human embryonic stem cell (hESC). The Hb expression profiles were also similar to hESC-derived RBCs; however, the β-globin expression was lower than the embryonic and fetal globins after fibroblasts transition to pluripotency status [124]. In 2016, Borger et al. showed that iPSCs that their HLA class I expression was silenced can effectively produce megakaryocyte (MK) and functional platelets with low immunogenicity. This is important because the most common cause of immunological platelet transfusion refractoriness is HLA-I-sensitive alloantibodies. Due to their ability to survive antibody-mediated complement and cell-dependent cytotoxicity, they can be used in thrombocytopenic patients with or without alloimmunity [125]. In 2014, Feng et al. described a rapid protocol for differentiation of hiPSC into megakaryocytes and functional platelets, resulting in the production of large amounts of cryopreservable MK progenitors. Hence, it gives the advantage of having countless platelets at a moment's notice upon thawing these cells [126].

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323324325326327328329330331

332

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334335336337338339340341

342343344345346347348349350351352353354355356

The unlimited proliferation of iPSC in-vitro not only provides desirable amounts of megakaryocyte and erythrocyte progenitors, but it could also generate other healthy cell lines, such as WBC. This could be used in lymphopenia or myelophthisic status in which the cell count and immune response are diminished. After receiving mature somatic cells from patients and reprogramming and differentiating them to HSC, first, the extracted HSCs could be differentiated to myeloid progenitors by IL-3, IL-1, GM-CSF, SCF, and IL-6. Then, some progenitors could be given megakaryocyte-stimulating cytokines like IL-3, hG-CSF, TPO, and SCF; and some could be given erythrocyte-stimulating cytokines like EPO, IL-3, hGM-CSF, and SCF depending on the purpose (Figure 1) [127].

Figure 1. iPS technology application in leukemia. First, somatic cells are reprogrammed to iPSC. The iHSCs are the first differentiated cells. Different cytokines drive differentiation to various immunologic cells like RBC, platelet, and malignant simulated cells

The autologous transplantation can be applied in non-severe cases using the aforementioned progenitors or hematopoietic products (RBC and PLT) without immunological rejection risk or other transmitted diseases. In urgent situations, pre-prepared heterologous products of iPSCs with the lowest expression of surface antigens can be used. Controllable expression of surface antigens in the iPSC-derived RBCs and PLTs enables a safe, heterologous transfusion. Evidently, using iPS technology in this field makes it possible to overcome many erythrocytopathies and thrombocytopathies in addition to leukemia.

Conclusion and Future Perspective

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As mentioned before, iPSC serving as a new invention in clinical field is an effective advancement in therapy and investigating the background of various diseases. Regarding new ideas and prospective projects, we have only scratched the surface of what we can do with it. In the future, advancement in research projects and suggestion of new theories will be observed in the treatment of many maladies like leukemia. The financial costs and mortalities will be reduced after routinely executing iPS technology in therapeutic clinics. Considered ideas and projects in this paper are in this direction. Besides leukemia, the mentioned iCTLs and iNK cells could be used extensively in the field of treatment by providing specificity against a particular pathogen. The invention of iPSC has also demonstrated promising results in different dimensions of the molecular biology of miRNA. The future world of iPSC will be significant and profound, and the power of imagination will propel this technology.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgement

We would like to thank Dr. Atashi and Dr. Azad for their valuable input on this article.

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