Induced pluripotent stem cells – developments on the road to big-time utilization

I have written about developments relating to cell reprogramming and induced pluripotent stem cells (iPSCs) in several previous blog posts, the most recent including A near-term application for iPSCs – making cell lines for drug testing, Induced pluripotent stem cells – second-rate stem cells so far, and Direct cell reprogramming.

Converting a scientific discovery into a practical technology always requires a number of secondary scientific discoveries and engineering developments –not basic breakthroughs but development of approaches that eliminate practical barriers and reduce costs.  It also requires mobilization of financial and business resources.  This blog post is concerned with three such current developments relating to iPSCs.

iPSCs from blood cells

It has long been known that practically any cell type can be reverted to iPSC status but, practically speaking, human iPSCs have almost always generated from fibroblasts.  Two of the barriers associated with producing  human iPSCs so far have therefore been 1.  The fibroblasts must be obtained from donors using a minor surgical procedure like a biopsy, limiting supply and raising cost, and 2.  The process of reversion of fibroblast cells to iPSCs takes a long time, around a month, and during this time mutations can accumulate.

Three new papers in the July 2 issue of Cell Stem Cell report on techniques for obtaining iPSCs from blood samples.  The blood can come from needle sticks, blood banks or umbilical cords of newborn. “In this issue of Cell Stem Cell, Staerk et al. (2010), Seki et al. (2010),  and Loh et al. (2010) each describe the derivation of human iPSCs from peripheral blood. Although seemingly incremental, this advance brings the stem cell field an important step closer to eventual clinical use.” 

The July 2010 paper Reprogramming of Human Peripheral Blood Cells to Induced Pluripotent Stem Cells explains the benefits of the process.  “The generation of patient-specific pluripotent cells is therefore an important goal of regenerative medicine. A major step to achieve this was the recent discovery that ectopic expression of defined transcription factors induces pluripotency in somatic cells (Lowry et al., 2008,Park et al., 2008b,Takahashi et al., 2007,Yu et al., 2007). Until now, the most common source from which to derive human iPSCs has been skin fibroblasts (Lowry et al., 2008,Park et al., 2008a,Park et al., 2008b,Takahashi et al., 2007,Yu et al., 2009). However, the requirement for skin biopsies and the need to expand fibroblast cells for several passages in vitro represent a hurdle that must be overcome to make iPSC technology broadly applicable. Peripheral blood can be utilized as an easily accessible source of patient tissue for reprogramming. Here we derived iPSCs from frozen human peripheral blood samples. Some of the iPSCs had rearrangements of the T cell receptor (TCR), indicating that T cells can be reprogrammed to pluripotency.– Our study demonstrates that peripheral blood can be utilized as an easily accessible source of patient tissue for reprogramming without the need to extensively maintain cell cultures prior to reprogramming experiments. This is an important step to make the iPSC technology more broadly applicable. Importantly, reprogramming of peripheral blood samples will permit access to numerous frozen samples already stored at blood banks. These samples are often of restricted use for research, because limited cell numbers do not allow experimental manipulations. This is particularly relevant if the patient is deceased and new material cannot be obtained. Generation of iPSCs from such samples could provide cell numbers large enough to retrospectively screen for genetic factors and to study molecular mechanisms underlying myeloid and lymphoid blood disorders.” 

The reversion process was not simple, however. “To increase the infection efficiency, we used a doxycycline-inducible lentivirus encoding all four factors Oct4, Klf4, Sox2, and c-Myc from a polycistronic expression cassette (pHAGE2-TetOminiCMV-hSTEMCCA) (Sommer et al., 2010). Blood cells were simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator (FUW-M2rtTA) (Hockemeyer et al., 2008) as well as the polycistronic vector. Infected blood cells were transferred onto feeder layers of mouse embryonic fibroblasts (MEFs) and cultured in the presence of IL-7 or G-CSF, GM-CSF, IL-6, and IL-3 and 2 g/ml doxycycline (Dox) for an additional 4 days (Figure 1A). At day 5 after Dox induction, the cells were transferred to human ESC medium containing 2 g/ml Dox, and 25 40 days later colonies were picked and expanded(ref).”

The second July 2010 paper in the same journal  issue Generation of Induced Pluripotent Stem Cells from Human Terminally Differentiated Circulating T Cells describes the approach used.  As in the case of the first paper, the iPSCs are mainly derived from blood T cells. “To avoid transgene integration during iPSC generation, we used an SeV vector, which is a minus-strand RNA virus that is not integrated into the host genome and is not pathogenic for humans (Li et al., 2000). We used a temperature-sensitive mutated SeV vector in these experiments to reduce transgene expression and SeV residue in generated lines. — To generate iPSCs from hTDCTCs, we used SeV to deliver multiple transgenes that encoded stem cell-specific transcription factors, such as OCT3/4, SOX2, KLF4, and c-MYC, into cells on day 6 of culture.”

The third paper in the same July 2010 issue of Cell Stem Cell, Reprogramming of T Cells from Human Peripheral Blood, reports “To test whether we can reprogram cells from routine peripheral blood (PB) sources, we obtained CD34⁺ purified blood samples from a healthy 49-year-old male donor who had undergone simple apheresis without cytokine priming. We also isolated mononuclear cells (PBMCs) from the peripheral blood samples collected by venipuncture of four healthy donors (28- to 49-years-old) via Ficoll density centrifugation. — To induce reprogramming of enriched CD34⁺ blood cells, we infected with lentiviruses expressing OCT4, SOX2, KLF4, and MYC reprogramming factors (Figure 1A). Colonies with well-defined hESC-like morphology were first observed 21 days after transduction (Figure 1B). For reprogramming of fresh peripheral blood mononuclear cells (PBMCs), we employed two rounds of lentiviral infection (day 0 and day 8) and isolated colonies with distinct flat and compact morphology with clear-cut round edges reminiscent of hESCs after a slightly longer latency of around 35 days (Figure 1C). With immunohistochemistry and flow cytometry, we analyzed the iPSC lines for expression of markers shared with hESCs. Consistent with their hESC-like morphology, both PB34 iPSCs and PBMC iPSCs stained positive for Tra-1-81, NANOG, OCT4, Tra-1-60, SSEA4, and alkaline phosphatase (AP) staining (Figures 1B 1D; Figures S1 A S1C available online; Chan et al., 2009).”

Reprogramming efficiency, already low for fibroblasts, is even worse using ordinary blood.  “We routinely observed a reprogramming efficiency of 0.002% for PB CD34⁺ cells (Table S1 ), comparable to prior experience with primary fibroblasts, mobilized PBMCs, and cord blood cell reprogramming (Takahashi et al., 2007,Park et al., 2008a,Loh et al., 2009,Haase et al., 2009). For PBMCs, we obtained hESC-like colonies at the lower efficiency of 0.0008% 0.001% (Table S1 )(ref).  This low reprogramming efficiency is yet-another problem that has to be solved before blood can routinely used instead of fibroblasts to produce iPSCs.

iPSCs and Mesenchymal-to-Epithelial Transition

Despite the fact that several groups of researchers have routinely been generating iPSCs for more than a couple of years now, exactly how the transcription factors like  OCT4, SOX2, c-MYC, and Klf4 work on a molecular basis to reset the epigenomic state of cells to a pluripotent basis has remained largely a mystery. Some light is shed on that issue by two papers published this month on the role of mesenchymal-to-epithelial transition (MET) in cell reprogramming.

One of the July 2010 papers is A Mesenchymal-to-Epithelial Transition Initiates and Is Required for the Nuclear Reprogramming of Mouse Fibroblasts. “Epithelial-to-mesenchymal transition (EMT) is a developmental process important for cell fate determination. Fibroblasts, a product of EMT, can be reset into induced pluripotent stem cells (iPSCs) via exogenous transcription factors but the underlying mechanism is unclear. Here we show that the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition (MET) orchestrated by suppressing pro-EMT signals from the culture medium and activating an epithelial program inside the cells. At the transcriptional level, Sox2/Oct4 suppress the EMT mediator Snail, c-Myc downregulates TGF-β1 and TGF-β receptor 2, and Klf4 induces epithelial genes including E-cadherin. Blocking MET impairs the reprogramming of fibroblasts whereas preventing EMT in epithelial cells cultured with serum can produce iPSCs without Klf4 and c-Myc. Our work not only establishes MET as a key cellular mechanism toward induced pluripotency, but also demonstrates iPSC generation as a cooperative process between the defined factors and the extracellular milieu.”

The other July 2010 paper is Functional Genomics Reveals a BMP-Driven Mesenchymal-to-Epithelial Transition in the Initiation of Somatic Cell Reprogramming. “Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expression of defined embryonic factors. However, little is known of the molecular mechanisms underlying the reprogramming process. Here we explore somatic cell reprogramming by exploiting a secondary mouse embryonic fibroblast model that forms iPSCs with high efficiency upon inducible expression of Oct4, Klf4, c-Myc, and Sox2. Temporal analysis of gene expression revealed that reprogramming is a multistep process that is characterized by initiation, maturation, and stabilization phases. Functional analysis by systematic RNAi screening further uncovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. We show that this is linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs that are key regulators of MET. These studies thus define a multistep mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming.” This paper indicates that expression profiling reveals three phases of cell reprogramming: an initiation phase where MET plays a major role, a maturation phase and a stabilization phase.  “ RNAi screening defines MET and BMP signaling as essential for reprogramming. BMP induces miR-200 family miRNAs to drive MET and somatic cell reprogramming(ref).”

Another-yet current paper that relates MET to Snail is Notch mediated epithelial to mesenchymal transformation is associated with increased expression of the Snail transcription factor.

Commercial exploitation of iPSCs to discover drugs

In the blog post A near-term application for iPSCs – making cell lines for drug testing I stated “There is another application for iPSCs which is likely to become very important in the immediate future: supplying large quantities of specialized body cells for research and drug testing purposes.”  Further, I discussed how a company called Cellular Dynamics International was setting out to produce human iPSC-derived cardiomyocytes on a substantial scale and market them for research and drug-testing purposes. 

Here I report on the activities of another new company, iPierian, which has just closed on $22 million of series b financing led by Google Ventures. “The company is developing and applying cellular reprogramming and differentiation technologies to harness the power of pluripotent stem cells to transform drug discovery and enable the promise of regenerative medicine. iPierian’s induced pluripotent stem (iPS) cell technology will advance the understanding of disease biology and make drug discovery and development faster and better informed. — iPierian plans to apply this revolutionary technology in its proprietary programs and leverage it in collaborations with pharmaceutical and biotechnology industry partners. The company’s reprogramming and differentiation technologies will enable the identification of promising new therapeutic candidates earlier in the drug discovery process. Through its efforts, iPierian expects to make pharmaceutical research more directly beneficial to patients and more cost-effective for its partners.”

iPierian is the result of the merger of two companies involving an impressive collection of individuals.  ”One, in Boston, was called Pierian, co-founded by George Daley, Doug Melton and Lee Rubin, a trio of stars from the Harvard Stem Cell Institute. The other, iZumi Bio, in Mountain View, CA, had the entrepreneurial leadership of Corey Goodman as chairman, and John Walker, a longtime director of stem cell pioneer Geron, as CEO. The merged operation boasts a blue chip crew of backers—Kleiner Perkins Caufield & Byers, Highland Capital, MPM Capital, and FinTech Global Capital, who have pumped in a combined $31.5 million(ref).”

“It’s more about using the power of stem cells to discover new drugs, so that iPierian can find treatments that no one ever could before because they were constrained by limited tools. iPierian has set its sights from the start on neurodegenerative diseases that really have no good treatments—like Lou Gehrig’s disease, Huntington’s disease, and eventually Parkinson’s and Alzheimer’s. The cells, with all their talent to morph into any kind of adult cell scientists want, won’t get injected into the patient to replace missing or damaged tissue. Instead, they could help the scientists gather new insights into these intractable diseases, speed up the development process, and save time. Then iPierian will use all of this information to make conventional small molecule pills, or injectable protein drugs. If it is successful, the patients wouldn’t even know it was because of stem cells(ref).”

“iPierian’s efforts are centered on applying its technology to diseases for which there are poor in vivo and in vitro models and limited therapeutic treatments to date. iPierian’s proprietary programs are initially focused on three neurological diseases, including Parkinson’s disease, spinal muscular atrophy and amyotrophic lateral sclerosis. These conditions have limited therapeutic treatments, and scientists have demonstrated the ability to differentiate the affected cell types in these disorders. — iPierian is also working on calcific aortic valve disease, through its collaboration with the Gladstone Institute of Cardiovascular Disease, and plans to expand further into cardiovascular and metabolic diseases through additional collaborations with pharmaceutical partners(ref).” 

The launching of iPierian is another example of how scientific, technical, economic and business factors are cooperating to ever-quicken the pace of discovery and make anti-aging breakthroughs more likely, a phenomenon I discussed in the blog post Factors that drive Giuliano’s Law.