Reprogramming peripheral blood mononuclear cells using an efficient feeder-free, non-integration method to generate iPS cells and the effect of immunophenotype and epigenetic state on HSPC fate
Abstract
Background and objectives In 2006 Shinya Yamanaka successfully reprogrammed
mouse fibroblasts back to an embryonic stem cell-like state (called induced
pluripotent cells, iPS cells) using retrovirus to introduce four genes that
encode critical transcription factor proteins (Oct4, Sox2, KLF4, and c-Myc). This
ability to reprogram has promising future applications in clinical and biomedical
research for study of diseases, development of candidate drugs and to support
therapeutic treatments in regenerative medicine. However, the clinical applications
have to meet GMP requirements without the risk of insertional mutagenesis
associated with retrovirus. Chromatin modifying agents are widely used in many
protocols to generate iPS cells and culture of blood CD34+ cells with chromatin-modifying
agents can lead to an increase in marrow repopulating cells and in the
case of valproic acid increased erythroid cell colony formation. We undertook
research to help understand what effects these reagents have on mobilised peripheral
blood (mPB) CD34+ cells and optimised the expansion medium protocol
to facilitate reprogramming work. This project aims to utilize peripheral blood
mononuclear cells (MNC), one of the most easily accessible tissues to generate iPS
cells using an efficient non-viral, feeder cell free methodology, with the ultimate
goal of moving this methodology towards clinical use.
Materials and Methods G-CSF mobilised peripheral blood, buffy coat, cord
blood and fetal liver were obtained from patients and donors under informed
consent and ethics committee approval. Haematopoietic stem/progenitor cells
CD34+ or CD133+) isolated by magnetic separation were flow cytometry sorted
into CD34+/CD133+, CD34+/CD133-, and CD34-/CD133+ sub-populations and
their lineage potential were assessed in colony forming unit assays. The effect
of epigenetic modifiers valproic acid and 5-aza-2-deoxycytidine used singly or in
combination with each other and with IL3 on phenotype and lineage potential
of cultured CD34+ cells from mobilised peripheral blood were assessed by flow
cytometry and colony-forming unit assays. Prior to reprogramming mononuclear cells from peripheral blood or CD34+
cells from blood were expanded in culture medium supplemented with stem
cell factor (SCF), Fms-related tyrosine kinase 3 ligand (Flt3L) and Interleukin-
3 (IL-3) for several days. Actively proliferating cells were reprogrammed by
electroporation using episomal vectors with an oriP/EBNA-1 backbone to deliver
five reprogramming genes, Oct4, Sox2, Lin28, L-Myc, and Klf4. Electroporated
cells were seeded onto matrigel coated plates immediately after transfection or
were reseeded after three days’ culture. Subsequently, cells were cultured in
specific medium on different days. When iPS colonies appeared, they were
picked and cultured as for ES cells. Once established, iPS cell lines were
immunophenotyped using flow cytometry and immunofluorescence and their
potential to differentiate into the three germ layers was assessed in vitro.
Results and Conclusion The largest subpopulation of CD34+ cells was
CD34+/CD133+ population which was essentially committed to myeloid colony
production, while much smaller CD34+/CD133- subpopulation had a greater capacity
to generate erythroid colonies. Optimised cytokine cocktail for expansion
of CD34+ cells included IL-3, important in improving expansion and maintaining
functionality of CD34+ cells. The optimised cytokine cocktail comprised 100
ng/ml SCF, 10 ng/ml Flt3L, and 20 ng/ml IL-3, which maintained CD34+ cells
and MNC in an active proliferating state. In addition, valproic acid and IL3 were
found to act synergistically, to increase the numbers of CD34+/CD36+ positive
cells. However, we found that an apparent increase in red cell colony formation
actually resulted from a decrease in white cell colonies, so no overall increase in
red cell colonies was seen when equivalent numbers of CD34+ cells were plated.
Proliferating MNC maintained in optimised cytokine cocktail were amenable
to electroporation for the effective delivery of episomal transcription factors
(Oct4, Sox2, Klf4, L-Myc, and Lin28) within a backbone of oriP/EBNA-1. We
successfully developed an efficient and simple method for reprogramming MNC
from fresh or frozen samples to generate induced pluripotent cells using episomal
vectors in a feeder-free system without any requirement for small molecules and
the highest reprogramming efficiency is 0.033% (65 colonies from 2 ◊ 105 seeding
MNC). The cytokine cocktail and reprogramming methods work better in CD34+
cells from cord blood or fetal liver, and we obtained 148 iPS colonies from 105
seeding cells (0.148%) at most. In addition, fibroblasts from adult and fetal liver
can be successfully reprogrammed using the same reprogramming method. The
use of episomal vectors with an oriP/EBNA-1 backbone to deliver reprogramming
genes, and efficient electroporation were the most important factors in efficiency
of the reprogramming process. In addition, it is pivotal to initiate transfection
when cells are actively proliferating. The iPS cell lines we generated maintained the successful expression of ES markers including Oct4, Nanog, SSEA3. SSEA4,
TRA-1-60 and TRA-1-81, and had the capacity to successfully differentiate into
cell types of ectoderm, mesoderm and endoderm layers in vitro.