Induced Pluripotent Stem Cells: Advances and Applications in Regenerative Medicine

Reprogramming adult somatic cells into induced pluripotent stem cells (iPSCs) through the ectopic expression of reprogramming factors offers truly personalized cell-based therapy options for numerous human diseases. The iPSC technology also provides a platform for disease modeling and new drug discoveries. Similar to embryonic stem cells, iPSCs can give rise to any cell type in the body and are amenable to genetic correction. These properties of iPSCs allow for the development of permanent corrective therapies for many currently incurable disorders. In this chapter, we summarize recent progress in the iPSC field with a focus on potential clinical applications of these cells

Impaired DNA replication within progenitor cell pools promotes leukemogenesis.

Impaired cell cycle progression can be paradoxically associated with increased rates of malignancies. Using retroviral transduction of bone marrow progenitors followed by transplantation into mice, we demonstrate that inhibition of hematopoietic progenitor cell proliferation impairs competition, promoting the expansion of progenitors that acquire oncogenic mutations which restore cell cycle progression. Conditions that impair DNA replication dramatically enhance the proliferative advantage provided by the expression of Bcr-Abl or mutant p53, which provide no apparent competitive advantage under conditions of healthy replication. Furthermore, for the Bcr-Abl oncogene the competitive advantage in contexts of impaired DNA replication dramatically increases leukemogenesis. Impaired replication within hematopoietic progenitor cell pools can select for oncogenic events and thereby promote leukemia, demonstrating the importance of replicative competence in the prevention of tumorigenesis. The demonstration that replication-impaired, poorly competitive progenitor cell pools can promote tumorigenesis provides a new rationale for links between tumorigenesis and common human conditions of impaired DNA replication such as dietary folate deficiency, chemotherapeutics targeting dNTP synthesis, and polymorphisms in genes important for DNA metabolism

Impaired DNA replication within progenitor cell pools promotes leukemogenesis.

Impaired cell cycle progression can be paradoxically associated with increased rates of malignancies. Using retroviral transduction of bone marrow progenitors followed by transplantation into mice, we demonstrate that inhibition of hematopoietic progenitor cell proliferation impairs competition, promoting the expansion of progenitors that acquire oncogenic mutations which restore cell cycle progression. Conditions that impair DNA replication dramatically enhance the proliferative advantage provided by the expression of Bcr-Abl or mutant p53, which provide no apparent competitive advantage under conditions of healthy replication. Furthermore, for the Bcr-Abl oncogene the competitive advantage in contexts of impaired DNA replication dramatically increases leukemogenesis. Impaired replication within hematopoietic progenitor cell pools can select for oncogenic events and thereby promote leukemia, demonstrating the importance of replicative competence in the prevention of tumorigenesis. The demonstration that replication-impaired, poorly competitive progenitor cell pools can promote tumorigenesis provides a new rationale for links between tumorigenesis and common human conditions of impaired DNA replication such as dietary folate deficiency, chemotherapeutics targeting dNTP synthesis, and polymorphisms in genes important for DNA metabolism

HU Treatment Enhances Bcr-Abl-Mediated Leukemogenesis

<div><p><i>Bcr-Abl</i> BMT recipients were generated and treated with HU as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030401#pbio-0030401-g006" target="_blank">Figure 6</a>.</p> <p>(A–B) Kaplan-Meier curves for vector (A), p190 <i>Bcr-Abl</i> (A), or p210 <i>Bcr-Abl</i> (B) transduced stem cell-transplanted mice with or without HU treatment are shown. The curves for p190 and p210 <i>Bcr-Abl</i> with HU are statistically different from the respective <i>Bcr-Abl</i>-untreated curves (<i>p</i> < 0.001 and <i>p</i> < 0.05, respectively). For (A) and (B), the average percent GFP⁺ in peripheral blood cells was 3%, and mice with similar percentages were segregated into the HU-treated and untreated groups. Mice were sacrificed when moribund, all with splenomegaly and massive increases in B220⁺GFP⁺ (for p190) or GR-1⁺GFP⁺ (for p210) cells in the spleen and peripheral blood.</p> <p>(C) Peripheral blood from morbid p190 <i>Bcr-Abl</i>-BMT mice (with or without HU, as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030401#pbio-0030401-g006" target="_blank">Figure 6</a>) or from a healthy vector BMT mouse was analyzed for B220 and GR-1 expression by flow cytometry.</p> <p>(D) BM cells from morbid p190 BMT mice were analyzed for the expression of c-Kit, CD34, CD43, B220, with side scatter and forward scatter (reflects cell size) as indicated. The dashed oval indicates the typical position of normal BM pre/pro-B-cells. All leukemic BM cells in untreated <i>Bcr-Abl</i> recipients expressed B220 and CD43 as shown in the upper panels. Leukemic BM cells from HU treated mice generally expressed less B220 (despite B220 expression in peripheral cells; see (A) and displayed more c-Kit⁺ and many more CD34⁺ cells. Percentages shown are averages (± SD) for three mice for each group. Two <i>Bcr-Abl</i>⁺ mice treated with HU that developed leukemia late (7 and 10 wk post-BMT) displayed an immunophenotype more similar to the <i>Bcr-Abl</i>-untreated mice.</p></div

HU Promotes the Competitive Expansion of <i>Bcr-Abl</i>⁺ Blood Progenitors Ex Vivo

<div><p>(A) BALB/c c-Kit⁺ BM cells were transduced with MSCV-<i>Bcr-Abl</i> or vector and cultured in stem cell medium with HU (0, 5, 10, 25, 50, or 100 μM) added starting at day 3. Flow cytometric analysis of GFP expression and total cell counting in cultures were performed every 3–4 d. The changes in GFP percentages in vector and Bcr-Abl cultures (upper graphs) and the cumulative expansion (log_e scale) of <i>Bcr-Abl</i>⁺ and vector cells (lower graphs) during HU treatment were plotted.</p> <p>(B) The percentages of cells expressing Bcr-Abl (day 13) as well as the mean fluorescence intensities (mfi) for the GFP⁺ population, which has been shown to reflect Bcr-Abl protein expression [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030401#pbio-0030401-b35" target="_blank">35</a>], are indicated. Most <i>Bcr-Abl</i>⁺ cells were c-Kit⁺CD34⁺, with or without HU treatment. HU treatment did not affect the intensity of GFP in vector cells (unpublished data).</p></div

E2f1/<i>E2f</i>2 Loss Promotes Bcr-Abl-Mediated Leukemias

<div><p>(A and B) <i>E2f1</i>⁺<i>2</i>⁺ or DKO mouse c-Kit⁺ cells were transduced with MSCV-<i>Bcr-Abl</i> and transplanted as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030401#pbio-0030401-g002" target="_blank">Figure 2</a>, except that in (B) a higher initial transduction efficiency was obtained (23.2% for <i>E2f1</i>⁺<i>2</i>⁺ and 24.1% for DKO). Initial transduction efficiencies for (A) were the same as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030401#pbio-0030401-g002" target="_blank">Figure 2</a>. Untransduced competitors were included as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030401#pbio-0030401-g002" target="_blank">Figure 2</a>, except that for the experiments shown in (B), competitors were c-Kit⁺-purified BM cells cultured in parallel to the transduced cultures but not infected, and each recipient mouse received 1.5 × 10⁵ untransduced c-Kit⁺ competitors combined with 1.4 × 10⁶<i>Bcr-Abl</i>-transduced progenitors. Kaplan-Meier curves are shown for both experiments. Mice were sacrificed when moribund, all with splenomegaly and lymphadenopathy. No mice transplanted with vector-expressing cells developed leukemia (unpublished data). The Kaplan-Meier curves for <i>Bcr-Abl</i>/DKO are statistically different from the <i>Bcr-Abl</i>/ <i>E2f1</i>⁺<i>2</i>⁺ (<i>p</i> < 0.001 for [A] and <i>p</i> < 0.0001 for [B]) and <i>Bcr-Abl</i>/DKO + <i>E2f1</i>⁺<i>2</i>⁺ competitors (<i>p</i> < 0.001 for [A], <i>p</i> < 0.02 for [B]) curves.</p> <p>(C) Peripheral blood, spleen, and BM from morbid leukemic <i>Bcr-Abl</i> BMT mice or from healthy vector BMT mice (from [B]) were analyzed for the expression of the indicated antigens and GFP by flow cytometry. Peripheral blood was analyzed 21 d post-BMT. The recipient of <i>Bcr-Abl</i>/DKO cells was sacrificed at 23 d and other recipients sacrificed at 29 d post-BMT. The mean fluorescence intensities (mfi) for peripheral B220⁺ cells are indicated.</p></div

Bcr-Abl Restores S Phase Progression in DKO Progenitors

<div><p>Wild-type or DKO donor mouse c-Kit⁺ cells were transduced with MSCV-<i>Bcr-Abl</i> or vector and transplanted as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030401#pbio-0030401-g002" target="_blank">Figure 2</a> but at higher efficiency.</p> <p>(A) Recipients were injected 3 wk post-transplant with BrdU and spleen B cell and myeloid progenitors (combined) isolated by flow sorting, separating GFP⁺ from GFP⁻. Immunofluorescence for BrdU together with PI staining for DNA content are shown for GFP⁻ cells (internal control) and GFP⁺ cells from <i>E2f1</i>⁺<i>2</i>⁺ or DKO recipients of <i>Bcr-Abl</i>-transduced stem cells. The percentages of cells with S phase DNA content but without significant BrdU incorporation (in the small square) are indicated.</p> <p>(B) The histograms shown in the insets in (A) plot the DNA content of cells gated as BrdU⁺, which are used to calculate the average lengths of S phase (from two experiments, ± SE).</p></div

The Inhibition of p53 Provides a Competitive Advantage for DKO, but Not Wild-Type, BM Progenitors

<div><p>(A) Wild-type or DKO BM cells were transduced with MSCV-DNp53, MSCV-DDp53, or vector, and transplanted into lethally irradiated BALB/c recipients. At 2 wk post-BMT, the percentage of GFP⁺ cells among peripheral blood GR-1⁺ cells was determined by flow cytometry. The contribution of GFP⁺ cells after 2 wk relative to the initial infection efficiency is shown. Initial infection efficiencies for each virus were similar for the two BM genotypes.</p> <p>(B) Genetic disruption of <i>p53</i> provides a proliferative advantage in the DKO but not wild-type background. The indicated mixtures of freshly harvested BM were used to reconstitute the hematopoietic system of lethally irradiated recipient BALB/c mice. At 6 wk post-BMT, the percentage of GFP⁺ nucleated cells in peripheral blood was determined by flow cytometry and fold expansion was determined relative to the initial mixture.</p></div