III. Abbreviations
1. Introduction
1.7. Current therapy of Type 1 diabetes
1.7.3. Cell–based therapy
The limitations of accessible islet sources as well as side effects of immunosuppression regimen bring a new idea: “cell–based therapy” in this field. Stem cells are the main source to reach insulin–producing cells (IPCs). However, different cell sources can be used to achieve this goal (figure 8). There are three distinct experimental methods for achieving this aim: overexpression of transcription factors, addition of chemical compounds like growth factors and cell–cell fusion or cell fusion [102, 103] in which, not terminally differentiated cells will be committed into IPCs [102].
Figure 8 adapted from [102]: Scheme of strategies to generate IPCs in vitro: A) Differentiation of pluripotent cells into IPC. B) Transdifferentiation of multipotent cells (MSCs) as well as somatic cells into IPC. C) The aim is the expansion of IPCs in the culture.
A
B C C
Page | 31 1.7.3.1. Stem cells
“The stem cell is the origin of life”.1 Stem cells are unspecialized cells that can unlimitedly proliferate (self–renewal character). Based on their origins, there are two main stem cells: embryonic stem cells (ESCs) (originated from Morula or Blastula stages), or adult stem cells (originated from each adult tissue like mesenchymal or hematopoietic bone marrow stem cells and adult–derived pancreatic stem cells) (figure 9) [104]. Based on their plasticity, stem cells are classified to three main categories: totipotent stem cells (can differentiate into all kinds of cells in the body plus extra–embryonic placenta), pluripotent stem cells (can differentiate into all kinds of cells in the body like ESCs and induced pluripotent stem cells (iPSC) and multipotent stem cells (can differentiate into several kinds of cells in the body like mesenchymal stromal cells (MSCs)). Although, high plasticity makes ESCs a powerful tool for regenerative medicine, they have some ethical concern (due to their embryo origin) as well as immunological rejection concern (due to difference in immunogenicity between ESCs donor and recipient) [104, 105]. Therefore, many scientists focused on pluripotent and multipotent stem cell studies to avoid these obstacles in ESCs.
Here, I review the two most commonly used “stem cells” in cell–based therapies:
iPSCs and bone marrow MSCs.
MSCs are mesenchymal cells, since they origin from the mesoderm and act as connective tissue. They are stromal cells because they form the supportive structure for the other cells in the tissue. Finally, they are multipotent cells, because they cam differentiate into various cell types, but they are not pluripotent, because they can not form an entire organ Of note, there is still a debate on whether MSCs should be called mesenchymal stem cells, or mesenchymal stromal cells,since they are not fully defined as stem cells and it is still a question mark whether they are able to transdifferentiate into nonmesenchymal cells [106].
1Stewart Sell
Page | 32 Figure 9 adapted from [104]: Human stem cells categories.
1.7.3.1.1. Induced pluripotent stem cells (iPSCs)
In 2006, Takahashi and Yamanaka first reported that adult mouse fibroblast cells reprogram into iPSCs via the overexpression of specific transcription factors [107].
This report made a new hope for making pluripotent cells from differentiated adult cells. IPSCs are an ideal source of pluripotent stem cells that are derived from any cell types and transplantable without immune rejection in autologous recipient. In parallel, individual patient derived–iPSCs are a useful tool for disease modeling in order to investigate cellular and molecular mechanisms of diseases.
Over the last few years, various range of cells reprogrammed via introducing a combination of transcription factors to make iPSCs. For instance, Stadtfeld et al.
Human Stem Cells
Adult
Infant
Fetal
Embryonic Germline
Somatic
Oogenia
Spermatogonia Wharton´s
Jelly Umbilical Cord blood
Abortus
(Fetal tissues) Fetal Stem cells
Gonadal ridge (6 weeks)
Blastocytes (5-7 days)
Embryonic Stem cells Embryonic Stem cells Umbilical cord Matrix Stem cells
Umbilical cord Blood Stem cells
Pancreas Gut
Eye
Neuronal
Epidermal (skin-hair)
Liver
Mesenchymal Bone marrow Stromal
Hemopoietic
Peripheral Blood
Bone marrow
Page | 33 introduced iPSCs derived pancreatic β–cells in 2008 [108]. IPSCs are generated by delivery of three/four genes or microRNA (miR) clusters (which are essential for maintenances of pluripotency and self–renewal such as Sox2, c–Myc, Klf4, Nanog, Oct3/4, Lin28 and/or miR302/367) to push somatic cell reprogramming [107, 109].
There are some critical issues regarding iPSCs. The first issue is the efficiency of iPSCs generation since higher number of efficient iPSCs potentially lead to higher number of efficient differentiated colonies. Different methods were applied for delivery of reprogramming factors into somatic cells which are plasmid [107], virus [110], miRNA [111], protein [112] and chemical compounds [113]. Plasmid transfection is an inefficient method (≤0.0002%) compared to viral infection (≤0.2%). Indeed, this efficiency is 10-fold lower than the same method applied for human fibroblast cells compared to mouse fibroblast cells [114]. Although non–integrated strategies were less efficient before, recent studies reported much improved non–integrated protocols [114, 115]. Rais, in 2013, reported that nearly 100% of somatic cells are reprogrammed to iPSCs via depletion of Mbd3 (a molecule that is involved in nucleosome remodeling and deacetylation) in both human and mouse in 5 days [115].
Furthermore, some other factors like cell types, origins and age directly affect the quality and efficiency of the reprogramming and differentiation stages [116].
Safety is the second important issue in using iPSCs for clinical applications. Chang and Sommer independently provided poly–cistronic lentiviral vectors in which all four reprogramming factors were introduced in a single lentiviral vector construct, since infection with a single lentiviral is safer than four vectors [110, 117]. Furthermore, safe transgene–free iPSCs were generated via a cocktail of novel molecules or chemical compounds such as histone methyltransferase inhibitors (BIX01294, BayK8644) [113], histone deacetylase inhibitors (valproic acid [118], butyrate [119]) or chromatin–
associated protein (Utf1) as well as anti–p53 specific siRNA combinations [111, 112].
Some of these inhibitors switch on epigenetic regulators like G9a histone methyltransferase inhibitor [113, 116, 120] and deacetylase inhibitors [116, 121, 122]
resulted in the high plasticity of iPSCs with the low quality of iPS–differentiated cells [123].
Page | 34 The third issue for using of iPSCs in clinics is immunogenicity. Recently, Araki and Guha independently found that transplantation of differentiated iPSCs to autologous recipients can be tolerated in some tissues due to negligible immunogenicity [124-126]. In another study, transplanted differentiated cells derived from iPSCs provoked immune response in a synergic mouse model due to abnormal gene expression in differentiated iPSCs, compared to ESCs [127].
The fourth major issue is tumorigenicity. Oncogenes such as C–Myc, Klf4 or the loss of tumor suppressor p53 are the oncogenic key factors to make iPSCs and they are being used to maintain survival and proliferation of iPSCs. It has been reported that simultaneous overexpression of C-Myc and KLf4 together with p53 knockdown resulted in a synergistic induction in reprogramming efficiency in fibroblasts-but all those are the factors which potentially make tumors [114, 128, 129]. iPSCs have been proposed but still have a long way to be clinically used in cell therapies.
1.7.3.1.2. Bone marrow mesenchymal stromal cells (MSCs)
For the first time, Friedenstein et al. introduced and characterized mouse bone marrow derived cells in 1966 [130]. They showed a group of bone marrow cells that have a potential to differentiate into multilineages and they are called “bone marrow mesenchymal stromal cells” based on their origins in bone marrow, fibroblast–shaped in the culture and no evidence of self–renewal properties in vivo [130]. Recent studies showed that MSCs can be found in the most of tissues such as skin, muscle, pancreas and adipose tissues [104, 131, 132]. Current studies also showed that MSCs have the capacity of self–renew in vivo [133, 134]. MSCs are easily accessible from the bone marrow. Plastic–adherence and colony forming unit–fibroblasts are the first MSCs characters in the culture. Bone marrow has approximately 0.01–0.001% MSCs that it is not enough for most research purposes [135]. However, these cells rapidly proliferate in the culture and their proliferation rate even accelerates using a platelet lysate instead of preselected FCS [136] or low oxygen tension due to the mimicry of their native microenvironment [137].
Page | 35 In 2006, the international society for cellular therapy has introduced minimal criteria to recognize MSCs by the positivity for surface antigens CD73 (identified by the MAb SH3 and SH4), CD90 and CD105 (identified by the MAb SH2) or the negativity for surface antigens CD34 (primitive hematopoietic progenitors marker), CD45 (pan–
leukocyte marker), CD19 and CD79a (B cells marker), CD14 and CD11b (monocytes and macrophages marker) and HLA class II markers as well as the possibility of differentiation into chondrogenic, osteogenic and adipogenic lineages [138].
MSCs circulate through the body via blood stream, migrate and home into injured tissues. Multiple studies showed that the transplantation of MSCs in injured mice improves their recovery [139-141] by the modulation of immune response as well as transdifferentiation (more evidence) or fusion (less evidence) with target cells in injured tissues [141, 142]. MSCs have not only the potential to transdifferentiate into different lineages in vitro [143, 144], but also, they can fuse with somatic cells in vitro as well as in vivo [145-147].
1.7.3.2. MSCs and immune system
MSCs have been proposed as an immunomodulator through direct mechanisms by cell–cell contact, or indirectly, by secretion of growth factors and cytokines [148]. The interaction between MSCs and immune cells affect both innate and adaptive immune response through inhibition of monocyte maturation, T/B lymphocytes proliferation and switching pro– to anti–inflammatory state by modulation of cytokine production [149].
Activated MSCs terminate proinflammatory signals by two mechanisms: 1) Activated MSCs change the phenotype of “killer” M1 macrophage to “healer” M2 macrophage via secretion of prostaglandin E2 (PGE2). 2) Activated MSCs produce a TNF–
stimulated gene 6 protein (TSG–6) that interacts with a glycoprotein (CD44) on the surface of M1 macrophages to decline TLR2/NF–kappaB signaling pathway results in reduction of proinflammatory molecules. The outcome is decreasing inflammation [150]. Additionally, the lack of donor antigens, low level of HLA class I and the absence of HLA class II make allogeneic MSCs a suitable source for transplantation [135, 151, 152].
Page | 36 1.7.3.3. MSCs in clinic
In 2002, Bartholomew et al. reported that the skin graft survives for a longer time by allogeneic MSCs injection in primates [152]. Later, Yuehua and coworkers showed that injected BMDCs into blastocyst could proliferate and differentiate into all organs in response to the tissue´s specific signals. Unlike ESCs, they did not observe any teratomas after intravenous MSC injection into immunodeficient mice [153]. Another report showed that BCM increase after MSC injection in STZ-induced diabetic mice [154]. Other studies showed that BMDCs circulate and home in different tissues after transplantation. For instance, this has been proven by detection of green fluorescent labeled BMDCs detected in pancreatic islets after injection of GFP+ bone marrow [155]. Co–transplantation of MSCs with different cells improves and prolongs the transplantation efficiency. For instance, transplantation of pancreatic islets with MSCs prolonged islet survival and improved their functions in a diabetic mouse model [156].
The impact of MSC co–transplantation with islets are summarized in figure 10. Further studies showed MSCs express a set of chemokine receptors such as CX3C receptor 1 (CX3CR1) and CXC receptor 12 (CXCR12), which are attracted by pancreatic islets by expression of CX3C ligand 1 (CX3CL1) and CXC ligand 12 (CXCL12) [157].
Therefore, MSCs would be an attractive vehicle for effective drug, gene or protein delivery to targeted cells [158]. For example, Wu et al. showed that co–transplantation of HGF+IL–IRa+ MSCs with islets improved the efficiency of islet transplantation [159].
Page | 37 Figure 10 adapted from [160]: The positive impact of MSCs on grafted islets in diabetic mouse model: the co–transplantation of MSCs and islets results in significantly improvement of islet transplantation via graft remodeling and immunomodulation.
Although, Melton and his colleagues showed that the proliferation of β–cells is the main source of new β–cells rather than cell differentiation [56], other studies showed the capacity of MSCs transdifferentiation into β–cells in vitro [161, 162] as well as in vivo [59, 163]. In summary, MSCs are important cells in clinics because of multiple advantages such as:
1- Source accessibility
2- Anti-inflammatory and, anti– apoptotic properties
3- Capability to differentiate into multilineages like adipocytes, neurons and pancreatic β–cells.
4- Lack of teratomas formation in vivo [164].
1.7.3.4. MSCs in regenerative medicine
Over the last three decades, many studies showed that MSCs are an important tool in regenerative medicine [142, 165]. Blau and her coworkers showed transplanted bone
Islets
MSC
Diabetic
mouse Graft remodeling
Immunomodulation d l
Page | 38 marrow cells which contain different cell types including MSCs (BMDC) contribute to generate myoblast cells in injured mice. In this process, BMDCs first acquire the muscle diploid stem cell fate called satellite cells and later they contribute to generate mature polyploid myofibers [166]. In another study, Hess et al. showed lower blood glucose levels in BMDCs injected nonobese diabetic mice; they reported more pancreatic β–cells in BMDCs injected mice suggesting that BMDCs indirectly transdifferentiate into vascular endothelial cells which resulted in higher rate of pancreatic progenitor cell proliferation [162, 163]. Indeed, PDX1–expressing MSCs could be transformed into E–cells and displayed insulin content, glucose–stimulated insulin secretion and reduced hyperglycemia in diabetic mice [165]. Prockop and his colleagues reported a sub-population of differentiated epithelium like cells in the mixture of injured epithelial cells and MSCs in the culture. This result confirmed this idea that some of MSCs transdifferentiated into epithelium like cells. Interestingly, they demonstrated that fusion was a frequent phenomenon and up to 1% of the MSCs were epithelial+ polyploid cells [142]. Therefore, it is possible that BMDCs transdifferentiate directly or indirectly after cell fusion with impaired cells in injured tissue in vivo.