Ruth K. Foreman

In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state

Nuclear transplantation can reprogramme a somatic genome back into an embryonic epigenetic state, and the reprogrammed nucleus can create a cloned animal or produce pluripotent embryonic stem cells. One potential use of the nuclear cloning approach is the derivation of ‘customized’ embryonic stem (ES) cells for patient-specific cell treatment, but technical and ethical considerations impede the therapeutic application of this technology. Reprogramming of fibroblasts to a pluripotent state can be induced in vitro through ectopic expression of the four transcription factors Oct4 (also called Oct3/4 or Pou5f1), Sox2, c-Myc and Klf4. Here we show that DNA methylation, gene expression and chromatin state of such induced reprogrammed stem cells are similar to those of ES cells. Notably, the cells—derived from mouse fibroblasts—can form viable chimaeras, can contribute to the germ line and can generate live late-term embryos when injected into tetraploid blastocysts. Our results show that the biological potency and epigenetic state of in-vitro-reprogrammed induced pluripotent stem cells are indistinguishable from those of ES cells.

Direct Reprogramming of Terminally Differentiated Mature B Lymphocytes To Pluripotency

Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency.Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-α (C/EBPα) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency.

Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells

Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs, we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first, followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene, marking fully reprogrammed cells, was only observed late in the process. Importantly, the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming.

Wnt Signaling Promotes Reprogramming of Somatic Cells to Pluripotency

Document S1. Supplemental Experimental Procedures, Supplemental References, One Table, and Three FiguresxDownload (.35 MB ) Document S1. Supplemental Experimental Procedures, Supplemental References, One Table, and Three Figures

Reprogramming of murine fibroblasts to induced pluripotent stem cells with chemical complementation of Klf4.

Ectopic expression of defined transcription factors can reprogram somatic cells to induced pluripotent stem (iPS) cells, but the utility of iPS cells is hampered by the use of viral delivery systems. Small molecules offer an alternative to replace virally transduced transcription factors with chemical signaling cues responsible for reprogramming. In this report we describe a small-molecule screening platform applied to identify compounds that functionally replace the reprogramming factor Klf4. A series of small-molecule scaffolds were identified that activate Nanog expression in mouse fibroblasts transduced with a subset of reprogramming factors lacking Klf4. Application of one such molecule, kenpaullone, in lieu of Klf4 gave rise to iPS cells that are indistinguishable from murine embryonic stem cells. This experimental platform can be used to screen large chemical libraries in search of novel compounds to replace the reprogramming factors that induce pluripotency. Ultimately, such compounds may provide mechanistic insight into the reprogramming process.

Pan-Src Family Kinase Inhibitors Replace Sox2 during the Direct Reprogramming of Somatic Cells **

Ectopic expression of the four transcription factors Oct4, Klf4, Sox2 and c-Myc reprograms adult somatic cells to induced pluripotent stem (iPS) cells.[1] Although iPS cells hold considerable promise as tools in research and drug discovery, the clinical application of iPS cells is hindered by the use of viruses that deliver the exogenous factors and modify the host genome. It is therefore of great interest to replace virally transduced factors with either proteins or small molecules. To date a number of compounds have been identified that facilitate reprogramming of somatic cells. Among these are kenpaullone[2], valproic acid[3] and inhibitors of TGFβ-signaling.[4] Here we have exploited a reporter based screen[2] to identify a new class of compounds that functionally replace Sox2: inhibitors of the Src family of kinases. These molecules provide novel tools to study the molecular mechanism of Sox2 in reprogramming. To screen for small molecule replacements of Sox2, mouse embryonic fibroblasts (MEFs) harboring the firefly luciferase (Fluc) gene in the Nanog locus[2] (NL-MEFs) were transduced with Oct4, Klf4 and c-Myc (OKM), seeded into 1536-well plates in standard growth media and assayed against a large chemical library[5] (750,000 compounds; 2.2 μM). Compounds that reproducibly and dose-dependently activated the NL reporter >2.5-fold over vehicle-treated controls (Figure 1a) were then counter-screened in a cell based SV40-driven Fluc assay to rule out false positives that directly and non-specifically induce luciferase signal.[2, 6] Figure 1 Chemical complementation of Sox2 To confirm that filtered hit compounds which activate Nanog gene expression also replace Sox2, iPS cell colony formation was used as a secondary assay. Specifically, Klf4 and c-Myc were delivered retrovirally to O4NR-MEFs[1b] (cells harboring a Doxycycline (Dox)-inducible Oct4 cDNA in the collagen locus and the neomycin-resistance gene in the Oct4 locus), and Oct4 expression was induced by addition of Dox to the culture media (day 0). Two days later, positive screen hits (1-10 μM) were added to OKM-expressing MEFs in place of Sox2. After 10 days of compound treatment, growth media was supplemented with neomycin to select for colonies that reactivated the endogenous Oct4 locus. The reactivation of epigenetically silenced pluripotency-associated genes is required for somatic cells to transition to the iPS cell state.[7] Dox-independent, neomycin resistant colonies were not observed in DMSO-treated (0.1%, v/v) controls, indicating that vehicle-treated cells had not removed the epigenetic silencing marks from the Oct4 promoter (which drives NeoR) and were thus not pluripotent. Among the compounds tested, one compound, iPYrazine (iPY; 10 μM), promoted the formation of neomycin-resistant iPS cell colonies (Figure 1b, blue bars) that survived and could be cultured in the absence of Dox. Transgenic Oct4 independent (minus Dox) growth of the iPY-treated iPS cells demonstrated that they had reactivated and relied on endogenous Oct4 to maintain the pluripotent state. In addition, OKM transduction combined with iPY treatment of MEFs carrying a GFP reporter under control of the endogenous Oct4 locus[8] also gave rise to stable, GFP-positive iPS cell lines (Figure S1, Supporting Information). iPS cells derived from O4NR-MEFs with iPY, Dox and KM-transduction grew as pluripotent stem cell colonies in the absence of Dox and iPY. Moreover, these cells were indistinguishable from ES cells by morphological criteria and expressed the pluripotency-associated markers Oct4 and SSEA1 (Figure 1c). We next tested the differentiation potential of the iPY-derived iPS cells in a teratoma assay by injecting 106 cells subcutaneously into NOD-SCID mice. Tumors were isolated 3 weeks later and histological analyses demonstrated that cell types of all three germ layers were present; these included neural tissues, bone, cartilage and ciliated epithelium (Figure 1d). Furthermore, iPY-derived iPS cells contributed to live chimeras, as shown in Figure 1d. The results from this series of analyses indicate that the iPY-derived, Sox2-free iPS cells are pluripotent. In order to identify the biological target of iPY, we profiled the compound against a biochemical panel of tyrosine kinases (51 kinases; Table S1). From this analysis, we found that iPY potently inhibited a number of tyrosine kinases at 5 μM. Commercially available inhibitors (Figures 2a-b and Table S2) of these candidate kinase targets were then assayed for their ability to replace Sox2 in the iPS cell colony formation assay. As shown in Figure 2b, the pan-Src family kinase (SFK) inhibitors Dasatinib[9] and PP1[10] (Figure 2b) were able to recapitulate the activity of iPY. Interestingly, both Dasatinib and PP1 were >2-fold more active than iPY and efficiently replaced Sox2 (Figure 2b). Moreover, the pan-SFK inhibitors gave rise to colonies with a similar efficiency to TGFβ inhibitors (SB-431542 and LY-364947). The latter have been reported to replace Sox2 and served as a positive control in this study.[4] In addition to TGFβ inhibitors, Ichida et al. have also reported that the SFK inhibitor PP1 is able to replace Sox2.[4a] Together with our work, these results indicate that iPY is likely playing a role in reprogramming by inhibiting Src kinases, although additional mechanisms cannot be excluded. Figure 2 Src family kinase and TGFβ-inhibitors recapitulate the Sox2 replacement activity of iPY SFKs are a class of proto-oncogene tyrosine kinases that include nine mammalian members (i.e., c-Src, Yes, Fyn, Fgr, Lck, Hck, Blk, Lyn and Frk).[11] Several members of the SFK family have been reported to influence embryonic stem (ES) cell self-renewal and differentiation.[12] For example, activation of c-Src signaling promotes ES cell differentiation.[13] Consistent with this observation we find that the activation of Src signaling in MEFs with JK239[14] potently inhibits 4-factor reprogramming (Figure 2c). Together, our results suggest that SFK signaling is an important mediator of somatic cell reprogramming, where activation of the SFK pathway prevents reprogramming and inhibition allows for reprogramming in the absence of exogenous Sox2. Previously, Ichida et al. demonstrated that small molecule mediated inhibition of TGFβ-signaling with LY-364947 or E-616452 can replace Sox2 through the activation of Nanog expression.[4a] The results from our screen, which rely on Nanog activation as a surrogate for the replacement of Sox2, suggest that the inhibition of SFK- and TGFβ-signaling may converge on a similar mechanism; that is, the function of Sox2 can be replaced during direct reprogramming by activating Nanog expression. Another potential scenario comes from the observation that both Nanog[15] and SFK inhibition[13] are capable of maintaining the self-renewing pluripotent state in ES cells. Thus, TGFβ inhibitor-mediated Nanog activation and pan-SFK inhibition may instead converge on a common mechanism in which the differentiation of newly formed iPS cells is prevented, thereby assisting in the transition to an undifferentiated state. In either case, it is interesting to note that inhibition of distinct signaling responses converge on a common end point. In summary, we applied a cell-based, high-throughput chemical screen to identify small molecules that replace Sox2 during somatic cell reprogramming. The identification of novel SFK inhibitors provides new chemical tools to study the mechanisms underlying direct reprogramming and may ultimately help to bring iPS cell technology one step closer to clinical application.

Case 14-2018: A 68-Year-Old Woman with a Rash, Hyponatremia, and Uveitis

A Woman with a Rash, Hyponatremia, and Uveitis A 68-year-old woman was admitted to the hospital because of a rash, hyponatremia, and anterior uveitis. The illness had begun 14 days earlier, during a trip to India. Diagnostic tests were performed.

A 92-Year-Old Male with Eosinophilic Asthma Presenting with Recurrent Palpable Purpuric Plaques

Churg-Strauss syndrome or eosinophilic granulomatosis with polyangiitis is a systemic vasculitis affecting the small and medium-sized vasculature. It is commonly associated with asthma and eosinophilia. Most patients are diagnosed at around the age of 40. We report a case of biopsy-confirmed Churg-Strauss syndrome in a 92-year-old male with a history of eosinophilic asthma and peripheral eosinophilia who was later diagnosed with Churg-Strauss syndrome.

Case 15-2017 — A 27-Year-Old Woman with Anemia, Thrombocytosis, and Skin Lesions after Travel Abroad

A 27-year-old woman presented with skin lesions that had developed after international travel. Biopsy of a skin lesion revealed granulomatous dermatitis. Fatigue, weakness, and pulselessness developed in the left arm. A diagnosis was made.

Lower extremity erythema and tightening in an elderly male

None. st: None declared. Drs Nambudiri and Angel contributed equally to this article. Correspondence to: Vinod E. Nambudiri, MD, MBA, Department of Dermatology, Brigham and Women’s Hospital, 221 Longwood Ave, Boston, MA 02115. E-mail: vnambudiri@partners.org. J Am Acad Dermatol 2017;76:e161-2. 0190-9622/