The disorders that are created by damaged tissues, dysfunctional organs and diseased cells can be treated by using replacement therapy using functional cells or tissues. The creation of human embryonic stem cell line was offered as a hope for patients that need replacement therapy. Patient specific ESCs would help in avoiding immune rejection by allogeneic transplantation. The patient specific ESCs were not being successfully created using somatic cell nuclear transfer technology (SCNT) until Yamanaka and his colleagues as well as Thompson in 2013 could generate them. ESC like stem cells called iPSCs (induced pluripotent stem cells) were created from somatic cells. Yamanaka identified four factors called Oct4, Sox2, Myc and Klf4, while the factors identified by Thompson were NANOG and LIN28.
Peripheral blood as a cellular source for reprogramming
Cellular reprogramming of fibroblasts or CD34+ blood cells into iPSCs is managed effectively by the factors like Oct4 and Sox2. Peripheral blood cells are considered as a reliable source for cellular reprogramming after studying about embryonic fibroblasts and dermal fibroblasts. Another report by Tao Cheng revealed that granulocytes in mouse have a higher reprogramming efficiency than hematopoietic stem cells as revealed by SCNT.
Most widely used cells in peripheral blood for reprogramming were mature T cells and primary progenitor cells. Several approaches were utilized in different laboratories for reprogramming of T cells into pluripotent cells. The stem cells in the blood will have a surface marker called CD34 and the population of CD34+ cells in blood is enhanced by magnetic activated cell sorting or MACS. The CD34+ MNCs cultured in cytokine supplement will expand the progenitors expressing CD71, CD36 and CD235a and were successfully reprogrammed into iPSCs.
Factors involved in reprogramming somatic cells into pluripotent cells
Mesendoderm specificity and neurectoderm specificity are managed by the expression of Oct4 and Sox2 factors respectively , which help in efficient reprogramming of CD34+ blood cells into pluripotent cells. Other studies found that GATA3, GATA6, and Sox7 are similar to Oct4 while GMNN is found similar to Sox2 factor used in reprogramming. Oct4 factor alone was found to be directly involved in reprogramming of CD34+ blood cells into MSCs while Sox2 alone was used to reprogram fibroblasts into NSCs. Klf4 is found to be a critical factor for inducing pluripotency.
BCL-XL is not a common factor used for reprogramming, while it was observed that BCL-XL factor helped in enhancing the Yamanaka factor mediated reprogramming of MNCs by 10-fold. Therefore, generation of integration free iPSCs from peripheral blood can be done by the factors like Oct4, Sox2, Klf4 and BCL-XL. Reprogramming efficiency is also enhanced by NANOG and LIN28 factors.
Methodology for reprogramming of blood cells into pluripotent cells
iPSC generation through lentiviral vectors
Viral vectors that were used for efficient reprogramming of fibroblasts were not found to be helpful in high level expression of reprogramming factors. Efficient reprogramming is carried out by the expression of reprogramming factors. Efficient cellular reprogramming depends on certain parameters while preparing an expression vector. These parameters are
• Post transcriptional regulatory element
• Synthetic genes
• Fusion with transactivation domain
• Stoichiometry of reprogramming factors
• Expression of multiple genes with IRES or dual promoters
• Expression of multiple genes using 2A sequences
Reprogramming efficiency is increased by 100 fold when the spleen focus forming viral promoter is embedded in the lentiviral vector. Transactivation domain (TAD) fused with reprogramming factors was found to increase reprogramming of MEFs by fifty fold as well as that of human and mouse fibroblasts. Efficient generation of iPSCs is done by balanced expression of four factors according to some studies. Cleavage due to ribosomal skipping during translation leads to generation of 2A sequences. These sequences were used for constructing polycistronic vectors to design for the expression of genes in equimolar levels.
Episomal vector (EV) is considered more advantageous than Sendai viral vector (SeV). Reprogramming of blood cells by EV is ten times more efficient than SeV. Generating EV plasmids are affordable and simple while SeV generation is expensive and more challenging.
The induced pluripotent stem cells provide large amounts of patient specific somatic cells for individual therapy and has capacity to rejuvenate cells from elder patients. The teratoma formation from undifferentiated iPSCs is observed as a large hurdle in the iPSC based therapy. Use of chemicals like quercetin could prevent successfully the formation of tumors from undifferentiated iPSCs.
The iPSCs derived from peripheral blood can be further differentiated into hepatocytes, MSCs and cardiomyocytes. Several studies have shown that many factors together are responsible for generating human HSCs from iPSCs.
The fibroblasts were directly reprogrammed into cardiomyocytes, hepatocytes, NSCs with the help of retroviral vectors, which express certain factors in the fibroblasts. Integration free iMSCs were also generated by EV vector.
Treatment of systemic disorders
Reprogramming of blood CD34+ cells In Vivo into MSCs or NSCs and muscle stem cells might cure the diseases like Duchene muscular dystrophy, skeletal dysplasia, Alzheimer's disease and amyotrophic lateral sclerosis which do not have any cure otherwise. Transient expression of the reprogramming factors through SeV and EV vectors in the blood cells can aid in the formation of somatic stem cells. The utility of SeV is restricted as this vector expresses virus specific factors that can trigger immune response to the cells that are transfected.
Xiao-Bing Zhang. Cellular reprogramming of human peripheral blood cells. Genomics Proteomics Bioinformatics 11 (2013). 264-274.
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