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Paravertebral extramedullary hematopoietic masses (EHMs) account for up to 15% of extramedullary pseudotumors in beta-thalassemia (BT) and are most likely related to compensatory hematopoiesis. In most cases, pseudotumors are incidentally detected, as the majority of patients are asymptomatic. Since June 2020, luspatercept is approved for the treatment of patients with BT who require regular red blood cell transfusions. Data addressing the safety and efficacy of luspatercept in patients with BT-associated EHMs are pending. To date (May 2022), paravertebral EHMs were observed in two asymptomatic patients out of currently 43 adult patients with BT registered at the Adult Hemoglobinopathy Outpatient Unit of the University Hospital Essen, Germany. In one of them, a paravertebral EHM was diagnosed more than 10 years prior to referral. Throughout observation time, treatment with luspatercept was associated with a clinically significant reduction in transfusion burden while allowing to maintain a baseline hemoglobin concentration of >= 10 g/dL aiming to suppress endogenous (ineffective) erythropoiesis associated with BT. Considering the rarity of paravertebral EHMs in BT, luspatercept might potentially represent a novel therapeutic option for these often-serious disease-associated complications. However, appropriate follow-up investigations are recommended to detect (early) treatment failures secondary to an undesired luspatercept-associated erythroid expansion.

Background Infection with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) may be life‐threatening, and specific antiviral drugs are currently not available. However, first studies indicated that convalescent plasma treatment might improve the clinical outcome of coronavirus disease 2019 (COVID‐19) patients. Study Design and Methods In the current study, we investigated the efficacy of convalescent plasma treatment in eight COVID‐19 patients. All the patients were critically ill, and seven of them were SARS‐CoV‐2 RNA–positive when starting treatment. SARS‐CoV‐2–specific antibodies were determined by an enzyme‐linked immunosorbent assay detecting immunoglobulin G (IgG) antibodies against the S1 protein (Euroimmun), and the neutralizing titers were determined with a cell‐culture‐based neutralization assay. Plasma treatment started between 4 and 23 days after the onset of symptoms. The patients were usually treated by three plasma units, each containing 200–280 ml, which was applied at day 1, 3, and 5. Results Donor sera had on average lower IgG antibody ratios and neutralizing titers than the COVID‐19 patients before the onset of treatment (median ratio of 5.8 and neutralizing titer of 1:320 vs. 7.5 and 1:640, respectively). Nevertheless, we observed an increase of antibody ratios in seven and of neutralizing titers in five patients after treatment; which did, however, not correlate with patient survival. Plasma treatment was effective in three patients, but five deceased despite treatment. Patients who deceased had a later treatment onset than survivors and finally died from multiple organ failure. Conclusion Our data indicate that the efficacy of convalescent plasma treatment of critically ill COVID‐19 patients who already had developed strong antiviral immune responses and organ complications is limited.

We investigated immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among a group of convalescent, potential blood donors in Germany who had PCR-confirmed SARS-CoV-2 infection. Sixty days after onset of symptoms, 13/78 (17%) study participants had borderline or negative results to an ELISA detecting IgG against the S1 protein of SARS-CoV-2. We analyzed participants with PCR-confirmed infection who had strong antibody responses (ratio >3) as positive controls and participants without symptoms of SARS-CoV-2 infection and without household contact with infected patients as negative controls. Using interferon-γ ELISpot, we observed that 78% of PCR-positive volunteers with undetectable antibodies showed T-cell immunity against SARS-CoV-2. We observed a similar frequency (80%) of T-cell immunity in convalescent donors with strong antibody responses but did not detect immunity in negative controls. We concluded that, in convalescent patients with undetectable SARS-CoV-2 IgG, immunity may be mediated through T cells. OA platinum

COVID-19 is a potential life-threatening viral disease caused by SARS-CoV-2 and was declared a pandemic by the WHO in March 2020. mRNA-based SARS-CoV-2 vaccines are routinely recommended in immune-compromised patients, including patients with AA, as these patients are at increased risk of contracting COVID-19 and developing a more severe course of disease. Between March 2021 and November 2021 relapse of AA occurred in four (age [median]: 53 years, range 30-84 years) out of 135 patients currently registered at our department and two de novo cases of AA in temporal context to vaccination against SARS-CoV-2, were documented. Median time after first COVID-19 vaccination and relapse of AA was 77 days. All relapsed patients were vaccinated with the mRNA-based vaccine Comirnaty (R). Relapse in two out of the four patients was refractory to CsA/eltrombopag, favoring IST with hATG/CsA or BMT, respectively. Our observations should prompt clinicians to take vaccine-induced relapse of AA or de novo AA after SARS-CoV-2 vaccination into account. Furthermore, careful clinical monitoring and vigilance for signs or symptoms that may indicate relapse of AA (e.g., bleeding complications) are indicated

The cellular response to SARS-CoV-2 vaccination and infection in allogeneic hematopoietic stem cell transplant (HSCT) recipients is not yet clear. In the current study, HSCT recipients prior to and post vaccination were tested for SARS-CoV-2-specific humoral and cellular immunity. Antibodies against spike (S) 1 were assessed by Anti-SARS-CoV-2 IgG ELISA (Euroimmun). Cellular immunity was analyzed by an in house interferon-gamma ELISpot and T-SPOT.COVID (Oxford Immunotec), using altogether seven SARS-CoV-2-specific antigens. In 117 HSCT patients vaccinated twice, SARS-CoV-2 IgG antibodies were significantly higher than in HSCT controls pre vaccination (p &lt, 0.0001). After the second vaccination, we observed a median antibody ratio of 4.7 and 68% positive results, whereas 35 healthy controls reached a median ratio of 9.0 and 100% positivity. ELISpot responses in patients were significantly (p &lt, 0.001) reduced to ≤33% of the controls. After the second vaccination, female HSCT patients and female healthy controls showed significantly higher antibody responses than males (6.0 vs. 2.1 and 9.2 vs. 8.2, respectively, p &lt, 0.05). Cellular immunity was diminished in patients irrespective of sex. In conclusion, especially male HSCT recipients showed impaired antibody responses after SARS-CoV-2 vaccination. Changing the vaccine schedule or composition could help increase vaccine responses.

ZusammenfassungÜber viele Dekaden hinweg war für die Behandlung hämatologischer Erkrankungen die einzige akzeptierte zelluläre Therapie die Transplantation von hämatopoetischen Stamm- und Progenitorzellpräparationen, autolog oder allogen, nach vorheriger Konditionierung als individualisierte Therapieform etabliert. Dabei impliziert diese Art der Therapie aus rechtlicher Sicht nur minimale Manipulationen der Transplantate (Zentrifugation, Konzentrierung, Verdünnung, immunmagnetische Anreicherung oder Depletionen, Kryokonservierung), sodass die Gewinnung, Verarbeitung, Qualitätskontrolle und Abgabe der Stammzell-/Immuntherapeutika zumeist komplett in der Hand transfusionsmedizinischer Institutionen liegen. Nach der Zulassung durch die Europäische Arzneimittelbehörde (EMA) im August 2018 sind erstmals kommerziell erhältliche, autologe chimäre Antigen-Rezeptor-T-Zellen (chimeric antigen-receptor-T-cells, CAR-T-cells) in Deutschland sowie einer Vielzahl anderer europäischer Länder für die individualisierte Therapie von Patienten mit ausgewählten malignen Neoplasien der B-Zell-Reihe verfügbar. Im Rahmen einer gemeinsamen Sitzung der Sektion „Stammzelltransplantation und Zelltherapie“ und der Sektion „Präparative und therapeutische Apherese“ der Deutschen Gesellschaft für Transfusionsmedizin und Immunhämatologie (DGTI) am 19.10.2018 in Frankfurt wurden mehrere Impulsvorträge zum Thema „Zugelassene CAR-T-Zell-Therapie ante portas: Rolle der Transfusionsmedizin“ gehalten. Dabei wurden die Themen Finanzierung und Kostenerstattung, regulatorische und rechtliche Rahmenbedingungen sowie verschiedene Aspekte der notwendigen Verantwortungsabgrenzung zwischen dem Hersteller des Ausgangsmaterials, dem pharmazeutischen Unternehmen und dem klinischen Anwender ausführlich diskutiert.

When patients with chronic kidney disease are infected with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) they can face two specific problems: virus‐specific immune responses may be impaired and remdesivir, an antiviral drug described to shorten recovery, is contraindicated. Antiviral treatment with convalescent plasma (CP) could be an alternative treatment option. In this case report, we present two kidney transplant recipients and two hemodialysis patients who were infected with SARS‐CoV‐2 and received CP. Antibodies against the receptor‐binding domain in the S1 subunit of the SARS‐CoV‐2 spike protein were determined sequentially by immunoglobulin G (IgG) enzyme‐linked immunosorbent assay (ELISA) and neutralization assay and specific cellular responses by interferon‐gamma ELISpot. Before treatment, in both kidney transplant recipients and one hemodialysis patient antibodies were undetectable by ELISA (ratio, Highlights After treatment with convalescent plasma we observed an increase of specific humoral and cellular immunity in two kidney transplant recipients and two haemodialysis patients with SARS‐CoV‐2 infection.However, the success of convalescent plasma therapy was only be temporary in one transplant recipient.Short‐term monitoring of viral load and antiviral immunity appears as mandatory for this patient group.

BackgroundWhen patients with chronic kidney disease are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) they can face two specific problems: Virus-specific immune responses may be impaired and remdesivir, an antiviral drug described to shorten the time to recovery, is contraindicated. Antiviral treatment with convalescent plasma could be an alternative treatment option.MethodsIn this case series we present two kidney transplant recipients and two patients dependent on haemodialysis who were infected with SARS-CoV-2 and received convalescent plasma. Antibodies against the spike 1 protein of SARS-CoV-2 were determined sequentially by IgG ELISA and neutralization assay and specific T cell responses by interferon-gamma ELISpot.ResultsPrior to treatment, in three patients antibodies were undetectable by ELISA (ratio < 1.1), corresponding to low neutralizing antibody titers (≤ 1:40). One patient was also negative to the ELISpot and two showed weak responses. After convalescent plasma treatment we observed an increase of SARS-CoV-2-specific antibodies (IgG ratio and neutralization titer) and of specific T cell responses. After intermittent clinical improvement one kidney transplant recipient again developed typical symptoms at day 12 after treatment and received a second cycle of convalescent plasma treatment. Altogether, three patients clinically improved and could be discharged from hospital. However, one multimorbid female in her early eighties deceased.ConclusionsOur data suggest that the success of convalescent plasma therapy may only be temporary in patients with chronic kidney disease; which requires an adaptation of the treatment regimen. Close monitoring after treatment is needed for this patient group.

Among the adult stem cells, multipotent mesenchymal stem cells (MSCs) turned out to be a promising option for cell-based therapies for the treatment of various diseases including autoimmune and cardiovascular disorders. MSCs bear a high proliferation and differentiation capability and exert immunomodulatory functions while being still clinically safe. As tissue-resident stem cells, MSCs can be isolated from various tissue including peripheral or umbilical cord blood, placenta, blood, fetal liver, lung, adipose tissue, and blood vessels, although the most commonly used source for MSCs is the bone marrow. However, the proportion of MSCs in primary isolates from adult tissue biopsies is rather low, and therefore MSCs must be intensively expanded in vitro before the MSCs find particular use in therapies that may require extensive and repetitive cell replacement. Therefore, more easily accessible sources of MSCs are needed. Here, we present a detailed protocol to generate tissue-typical MSCs by direct linage conversion using transcription factors defining target MSC identity from murine induced pluripotent stem cells (iPSCs).

In vertebrates, the cellular compartment of blood is composed of many different cell types, including lymphocytes (T- and B-cells), Natural Killer (NK) cells, myeloid cells (granulocytes and monocytes/macrophages), erythrocytes (red blood cells), platelets (derived from megakaryocytes) and mast cells. Each day, humans need approximately 100 billion of those cells to be produced. Ultimately, all are the progeny of multipotent hematopoietic stem cells (HSCs), the central organizers of the hierarchical process of blood cell formation (hematopoiesis) [reviewed by (Orkin and Zon Cell 132:631–44, 2008)]. Because of their unique capability to home to the bone marrow, expand their numbers by self-renewal divisions and reconstitute life-long hematopoiesis after transplantation, they are the key for the cure a number of hematologic diseases with one single treatment.

Among the adult stem cells, multipotent mesenchymal stem cells (MSCs) turned out to be a promising option for cell-based therapies for the treatment of various diseases including autoimmune and cardiovascular disorders. MSCs bear a high proliferation and differentiation capability and exert immunomodulatory functions while being still clinically safe. As tissue-resident stem cells, MSCs can be isolated from various tissue including peripheral or umbilical cord blood, placenta, blood, fetal liver, lung, adipose tissue, and blood vessels, although the most commonly used source for MSCs is the bone marrow. However, the proportion of MSCs in primary isolates from adult tissue biopsies is rather low, and therefore MSCs must be intensively expanded in vitro before the MSCs find particular use in therapies that may require extensive and repetitive cell replacement. Therefore, more easily accessible sources of MSCs are needed. Here, we present a detailed protocol to generate tissue-typical MSCs by direct linage conversion using transcription factors defining target MSC identity from murine induced pluripotent stem cells (iPSCs).

Long-term ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells (HSPCs) proves to be unfeasible as cellular differentiation occurs when HSPCs are detached from their supporting bone marrow stem cell niche. This issue renders it difficult to make use of the proliferation capacity of HSPCs to subsequently produce functional blood cells in relevant numbers, e.g. for cell therapy approaches. To circumvent this challenge, leukemia-associated chimeric transcription factors, including MLL fusion proteins, can be exploited for their pronounced ability to propel cell proliferation while preserving cell immaturity. By designing the protein's activity controllable, the immature state can be abolished at an arbitrary point in time enabling terminal differentiation. In this study, we employed the fusion gene mixed lineage leukemia/eleven nineteen leukemia (MLL-ENL) for engineering an inducible protein switch. For this purpose, we fused the coding sequence of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the transcription factor MLL-ENL and subsequently expressed the protein switch (DD-MLL-ENL) in human CD34+ HSPCs derived from adult healthy donors. In the presence of the specific ligand Shield1, DD-mediated protein degradation is prevented leading to massive and long-term expansion of HSPC-derived late monocytic precursors in the presence of IL-3, IL-6, SCF, FLT3-L, TPO and GM-CSF. The cells do not exhibit additional driver mutations, feature a normal karyotype and telomere length, and sustain immaturity that is strictly dependent on Shield1 supplementation every other day even after two years of ex vivo culture. Upon Shield1 deprivation, the cells completely lost self-renewal and colony-forming properties and spontaneously differentiated. By changing the cytokines to GM-CSF in combination with IFN-γ and LPS we differentiated the progenitor cells into macrophages (MΦ) (Fig. 1 A, B). Immunophenotypic characterization revealed upregulation of the monocyte/macrophage-associated surface markers CD14, CD80, CD86, CD163 and MHC class I and II, concordant with monocytic morphology as judged by cytospin preparations. Analysis of the transcription of selected inflammatory genes, including IL-6 and IL-10, revealed overlapping M1 and M2 macrophage characteristics. Furthermore, mRNA expression profiles using nCounter Systems technology covering a total of 770 myeloid innate immunity-related genes proves the cells' identity as differentiated phagocytes shown by upregulation of gene clusters involved in Fc receptor signaling, TLR signaling, antigen presentation and T cell activation. In functional assays, we demonstrated the ability of the obtained cells to migrate towards the chemokine CCL2 in a 3D chemotaxis assay, attach to VCAM-1 under flow and shear stress and produce reactive oxygen species. Regarding the cells' phagocytic capability, we could verify the uptake of bacterial particles as well as apoptotic cells in efferocytosis assays. Finally, we demonstrated IgG Fc region recognition and binding by the expressed Fcγ receptors enabling phagocytosis of lymphoblastic tumor cells, including Daudi, Raji and patient-derived MCL cells in an antibody-dependent manner using rituximab (RTX), daratumumab (Dara) and trastuzumab (Trast) as a negative control (Fig. 1C). Overall, we could demonstrate the conversion of a harmful leukemic transcription factor into a useful molecular tool for large-scale ex vivo production of functional blood cells. Such engineered controllable protein switches might have the potential to be employed as molecular tools to produce functional immune cells for cell-based immunotherapeutic approaches. Figure 1 Figure 1. Disclosures Redondo Monte: Minaris Regenerative Medicine: Current Employment. Beier: Alexion: Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Other: Travel reembursement. Weigert: Janssen: Speakers Bureau; Epizyme: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding. Greif: AstraZeneca: Honoraria.

Background: The de novo generation of patient-specific hematopoietic stem and progenitor cells from induced pluripotent stem cells (iPSCs) has become a promising approach for cell replacement therapies in the future. However, efficient differentiation protocols for producing fully functional human hematopoietic stem cells are still missing. In the mouse model, ectopic expression of the human homeotic selector protein HOXB4 has been shown to enforce the development of hematopoietic stem cells (HSCs) in differentiating pluripotent stem cell cultures. However, the mechanism how HOXB4 mediates the formation of HSCs capable of long-term, multilineage repopulation after transplantation is not well understood yet. Methods: Using a mouse embryonic stem (ES) cell-based differentiation model, we asked whether retrovirally expressed HOXB4 induces the expression of Runx1/AML1, a gene whose expression is absolutely necessary for the formation of definitive, adult HSCs during embryonic development. Results: During ES cell differentiation, basal expression of Runx1 was observed in all cultures, irrespective of ectopic HOXB4 expression. However, only in those cultures ectopically expressing HOXB4, substantial amounts of hematopoietic progenitors were generated which exclusively displayed increased Runx1 expression. Conclusions: Our results strongly suggest that HOXB4 does not induce basal Runx1 expression but, instead, mediates an increase of Runx1 expression which appears to be a prerequisite for the formation of hematopoietic stem and progenitor cells.

Cell-based therapies using adult stem cells are promising options for the treatment of a number of diseases including autoimmune and cardiovascular disorders. Among these, vascular wall-derived mesenchymal stem cells (VW-MSCs) might be particularly well suited for the protection and curative treatment of vascular damage because of their tissue-specific action. Here we report a novel method for the direct conversion of human skin fibroblasts towards MSCs using a VW-MSC-specific gene code (HOXB7, HOXC6 and HOXC8) that directs cell fate conversion bypassing pluripotency. This direct programming approach using either a self-inactivating (SIN) lentiviral vector expressing the VW-MSC-specific HOX-code or a tetracycline-controlled Tet-On system for doxycycline-inducible gene expressions of HOXB7, HOXC6 and HOXC8 successfully mediated the generation of VW-typical MSCs with classical MSC characteristics in vitro and in vivo. The induced VW-MSCs (iVW-MSCs) fulfilled all criteria of MSCs as defined by the International Society for Cellular Therapy (ISCT). In terms of multipotency and clonogenicity, which are important specific properties to discriminate MSCs from fibroblasts, iVW-MSCs behaved like primary ex vivo isolated VW-MSCs and shared similar molecular and DNA methylation signatures. With respect to their therapeutic potential, these cells suppressed lymphocyte proliferation in vitro, and protected mice against vascular damage in a mouse model of radiation-induced pneumopathy in vivo, as well as ex vivo cultured human lung tissue. The feasibility to obtain patient-specific VW-MSCs from fibroblasts in large amounts by a direct conversion into induced VW-MSCs could potentially open avenues towards novel, MSC-based therapies.

Scientific Reports 6: Article number: 30792; published online: 03 August 2016; updated: 08 March 2018 This Article contains typographical errors. In the Results section, “We reprogrammed primary dermal fibroblasts isolated from a female patient with AS harboring a three-base pair deletion in exon 4 of the UBE3A gene (accession NM_130838)11, and from a normal healthy control person.

The transcription factor RUNX1 is a master regulator of normal hematopoiesis and is involved in cell fate decisions. RUNX1 mutations have been shown to contribute to the development of myeloid neoplasms, and in myelodysplastic syndromes (MDS) it is one of the most frequently mutated genes. Such mutations lead to RUNX1 proteins that lack transactivation activity or DNA-binding ability resulting in a loss of its tumor suppressor function. The dominant-negative short isoform RUNX1a resembles truncated RUNX1 mutants and inhibits the function of the full-length RUNX1 proteins. Additionally, a recently published study identified overexpression of RUNX1a but not full-length RUNX1 in CD34+-cells from patients with myelodysplastic/myeloproliferative disease, which increased with disease progression. This strongly suggests that truncated RUNX1 plays a pivotal role in myelodysplastic disease. However, the precise molecular functions of mutating RUNX1, particularly with respect to the identity of RUNX1 target genes conferring its tumor suppressor function, remain unclear. Previously, our group reported that overexpression of RUNX1a immortalized murine hematopoietic stem and progenitor cells (HSPCs) in vitro. Immunophenotyping of these cells confirmed the expansion of an immature subpopulation defined as Lin- Sca1+ Kit+ (LSK). This phenotype was reversed upon turning RUNX1a-expression off and led to a loss of Sca1 expression (Lin- Kit+, LK). To further understand the molecular consequences of RUNX1a overexpression we sorted the LK cells and LSK cells before and 36h after RUNX1a-expression was turned off. Next, we performed microarray analysis to assess differential gene expression in these different subpopulations. Gene set enrichment analysis (GSEA) identified upregulation of genes highly expressed in hematopoietic stem cells (HSC) and leukemic stem cells (LSCs) in RUNX1a-expressing LSK cells compared to those LSK cells in which RUNX1a expression was turned off. Conversely, a gene signature associated with stemness and self-renewal was lost in LK-cells when RUNX1a expression was turned off. Among the eleven leading edge genes, we found genes implicated in leukemogenesis, stem cell regulation, or both such as Erg, Meis1 and Bcl11a. To further understand the role of RUNX1a in vivo we transplanted C57Bl6 mice (n=29) with HSPCs expressing RUNX1a in a competitive reconstitution setting. Consistent with the immortalization of HSPCs in vitro, RUNX1a-overexpressing HSPCs expanded in the bone marrow of transplanted mice. We observed significantly higher frequencies of LK (2.9-fold) and LSK cells (5-fold) in the RUNX1a-expressing bone marrow cells compared to transplanted control mice. High frequencies of RUNX1a-expressing cells in the bone marrow were associated with lower frequencies of RUNX1a-expressing cells in the peripheral blood indicating a differentiation block. In addition, we found that 85% of the RUNX1a-expressing cells were committed to the myeloid lineage (CD11b+/Ly6G+) at the expense of the lymphoid lineage (B220 and CD3e). Moreover, RUNX1a expression led to an increased percentage (65%) of immature erythroblasts (Ter119-) in the bone marrow compared to control cells (55%). In summary, we have demonstrated that RUNX1a overexpression immortalized HSPCs by upregulation of genes involved in leukemogenesis, stemness and self-renewal. In vivo such HSPCs showed a competitive advantage that was associated with a block of differentiation. Our study, particularly the gene expression analysis, provides novel insights into genetic drivers contributing to the development of myeloid malignancies in patients with RUNX1 mutations. Disclosures No relevant conflicts of interest to declare.

The de novo generation of patient-specific hematopoietic stem and progenitor cells from induced pluripotent stem cells (iPSCs) has become a promising approach for cell replacement therapies in the future. However, efficient differentiation protocols for producing fully functional human hematopoietic stem cells are still missing. In the mouse model, ectopic expression of the human homeotic selector protein HOXB4 has been shown to enforce the development of hematopoietic stem cells (HSCs) in differentiating pluripotent stem cell cultures. However, the mechanism how HOXB4 mediates the formation of HSCs capable of long-term, multilineage repopulation after transplantation is not well understood yet.Using a mouse embryonic stem (ES) cell-based differentiation model, we asked whether retrovirally expressed HOXB4 induces the expression ofDuring ES cell differentiation, basal expression ofOur results strongly suggest that HOXB4 does not induce basal

Summary The vascular wall (VW) serves as a niche for mesenchymal stem cells (MSCs). In general, tissue-specific stem cells differentiate mainly to the tissue type from which they derive, indicating that there is a certain code or priming within the cells as determined by the tissue of origin. Here we report the in vitro generation of VW-typical MSCs from induced pluripotent stem cells (iPSCs), based on a VW-MSC-specific gene code. Using a lentiviral vector expressing the so-called Yamanaka factors, we reprogrammed tail dermal fibroblasts from transgenic mice containing the GFP gene integrated into the Nestin-locus (NEST-iPSCs) to facilitate lineage tracing after subsequent MSC differentiation. A lentiviral vector expressing a small set of recently identified human VW-MSC-specific HOX genes then induced MSC differentiation. This direct programming approach successfully mediated the generation of VW-typical MSCs with classical MSC characteristics, both in vitro and in vivo., Highlights • In vitro generation of (VW)-typical MSCs from iPSCs based on a specific HOX code • Reprogrammed fibroblasts (NEST-iPSCs) facilitated lineage tracing • A lentiviral vector expressing HOXB7, HOXC6, and HOXC8 induced MSC differentiation • Generated VW-MSCs showed classical MSC characteristics in vitro and in vivo, In this article, Klein and colleagues show that iPSCs generated from skin fibroblasts of transgenic mice carrying a GFP gene under the control of the endogenous Nestin promoter to facilitate lineage tracing (NEST-iPSCs) can be directly programmed toward mouse vascular wall-typical multipotent mesenchymal stem cells (VW-MSC) by ectopic lentiviral expression of a previously defined VW-MSC-specific HOX code.

Pluripotent stem cells hold great promise for future applications in many areas of regenerative medicine. Their defining property of differentiation towards any of the three germ layers and all derivatives thereof, including somatic stem cells, explains the special interest of the biomedical community in this cell type. In this review, we focus on the current state of directed differentiation of pluripotent stem cells towards hematopoietic stem cells (HSCs). HSCs are especially interesting because they are the longest known and, thus, most intensively investigated somatic stem cells. They were the first stem cells successfully used for regenerative purposes in clinical human medicine, namely in bone marrow transplantation, and also the first stem cells to be genetically altered for the first successful gene therapy trial in humans. However, because of the technical difficulties associated with this rare type of cell, such as the current incapability of prospective isolation, in vitro expansion and gene repair by homologous recombination, there is great interest in using pluripotent stem cells, such as Embryonic Stem (ES-) cells, as a source for generating and genetically altering HSCs, ex vivo. This has been hampered by ethical concerns associated with the use of human ES-cells. However, since Shinya Yamanaka´s successful attempts to reprogram somatic cells of mice and men to an ES-cell like state, so-called induced pluripotent stem (iPS) cells, this field of research has experienced a huge boost. In this brief review, we will reflect on the status quo of directed hematopoietic differentiation of human and mouse pluripotent stem cells.

Summary Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for regenerative therapies. Primarily, this has been achieved in mouse cells by overexpression of the homeotic selector protein HOXB4. The exact cellular stage at which HOXB4 promotes hematopoietic development, in vitro, is not yet known. However, its identification is a prerequisite to unambiguously identify the molecular circuits controlling hematopoiesis, since the activity of HOX proteins is highly cell and context dependent. To identify that stage, we retrovirally expressed HOXB4 in differentiating mouse embryonic stem cells (ESCs). Through the use of Runx1(−/−) ESCs containing a doxycycline-inducible Runx1 coding sequence, we uncovered that HOXB4 promoted the formation of hemogenic endothelium cells without altering endothelial cell development. Whole-transcriptome analysis revealed that its expression mediated the upregulation of transcription of core transcription factors necessary for hematopoiesis, culminating in the formation of blood progenitors upon initiation of Runx1 expression., Highlights • HOXB4 induces hemogenic endothelium formation from differentiating ESCs • Hematopoiesis is not promoted at the cost of endothelial cell development • HOXB4 induces a transcriptional program closely resembling dorsal aorta HE cells, In this article, Klump and colleagues demonstrate that the human homeotic selector protein HOXB4 promotes ESC-derived hematopoiesis by inducing hemogenic endothelium formation, in vitro. It propels hematopoietic specification by upregulating the transcription of genes essential for hematopoietic development, such as those encoding members of the so-called heptad transcription factors.

Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin-specific gene expression that is regulated by a differentially methylated region. Gene mutations or failures in the imprinting process lead to the development of imprinting disorders, such as Angelman syndrome. The symptoms of Angelman syndrome are caused by the absence of functional UBE3A protein in neurons of the brain. To create a human neuronal model for Angelman syndrome, we reprogrammed dermal fibroblasts of a patient carrying a defined three-base pair deletion in UBE3A into induced pluripotent stem cells (iPSCs). In these iPSCs, both parental alleles are present, distinguishable by the mutation and express UBE3A. Detailed characterization of these iPSCs demonstrated their pluripotency and exceptional stability of the differentially methylated region regulating imprinted UBE3A expression. We observed strong induction of SNHG14 and silencing of paternal UBE3A expression only late during neuronal differentiation, in vitro. This new Angelman syndrome iPSC line allows to study imprinted gene regulation on both parental alleles and to dissect molecular pathways affected by the absence of UBE3A protein.

The hepatocyte NF (HNF) family of transcription factors regulates the complex gene networks involved in lipid, carbohydrate, and protein metabolism. In humans, HNF1A mutations cause maturity onset of diabetes in the young type 3, whereas murine HNF6 participates in fetal liver B lymphopoiesis. In this study, we have identified a crucial role for the prototypical member of the family HNF1A in adult bone marrow B lymphopoiesis. HNF1A−/− mice exhibited a clear reduction in total blood and splenic B cells and a further pronounced one in transitional B cells. In HNF1A−/− bone marrow, all B cell progenitors—from pre-pro–/early pro–B cells to immature B cells—were dramatically reduced and their proliferation rate suppressed. IL-7 administration in vivo failed to boost B cell development in HNF1A−/− mice, whereas IL-7 stimulation of HNF1A−/− B cell progenitors in vitro revealed a marked impairment in STAT5 phosphorylation. The B cell differentiation potential of HNF1A−/− common lymphoid progenitors was severely impaired in vitro, and the expression of the B lymphopoiesis-promoting transcription factors E2A, EBF1, Pax5, and Bach2 was reduced in B cell progenitors in vivo. HNF1A−/− bone marrow chimera featured a dramatic defect in B lymphopoiesis recapitulating that of global HNF1A deficiency. The HNF1A−/− lymphopoiesis defect was confined to B cells as T lymphopoiesis was unaffected, and bone marrow common lymphoid progenitors and hematopoietic stem cells were even increased. Our data demonstrate that HNF1A is an important cell-intrinsic transcription factor in adult B lymphopoiesis and suggest the IL-7R/STAT5 module to be causally involved in mediating its function.

Genetic manipulation of hematopoietic stem and progenitor cells is an important tool for experimental and clinical applied hematology. However, techniques that allow for gene targeting, subsequent in vitro selection, and expansion of genetically defined clones are available only for ES cells. Such molecularly defined and, hence, “safe” clones would be highly desirable for somatic gene therapy. Here, we demonstrate that in vitro differentiated ES cells completely recapitulate the growth and differentiation properties of adult bone marrow cells, in vitro and in vivo , when ectopically expressing HOXB4. Myeloid development was enforced and (T) lymphoid development suppressed over a wide range of expression levels, whereas only high expression levels of the transcription factor were detrimental for erythroid development. This indicates a close association between the amounts of ectopic HOXB4 present within a progenitor cell and and the decision to self renew or differentiate. Because HOXB4 mediates similar fates of ES-derived and bone marrow hematopoietic stem cells, the primitive embryonic cells can be considered a promising alternative for investigating hematopoietic reconstitution, in vivo , based on well defined clones. Provided that HOXB4 levels are kept within a certain therapeutic window, ES cells also carry the potential of efficient and safe somatic gene therapy.

The homeodomain transcription factor HOXB4 is one of the most attractive tools to expand hematopoietic stem cells in vitro and in vivo and to promote the formation of hematopoietic cells from in vitro differentiated embryonic stem cells. However, the expression levels compatible with the favorable effect of enhanced self-renewal without perturbing differentiation, in vivo, remain to be determined. In this paper, we discuss the necessity to define the "therapeutic width" of HOXB4 expression, based on observations from our lab and others that demonstrate that ectopic HOXB4 expression leads to a concentration-dependent perturbation of lineage differentiation of mouse and human hematopoietic cells. In summary, the combined results argue in favor of the existence of certain threshold levels for HOXB4 activity that control the differentiation and self-renewal behavior of hematopoietic stem and progenitor cells. Indeed, existing evidence suggests that dosage effects of ectopically expressed transcription factors may be more the rule than an exception.

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice or in competition with control vector–transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P = .01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P

Differentiation of pluripotent embryonic stem (ES) cells can recapitulate many aspects of hematopoiesis, in vitro, and can even generate cells capable of long-term multilineage repopulation after transplantation into recipient mice, when the homeodomain transcription factor HOXB4 is ectopically expressed. Thus, the ES-cell differentiation system is of great value for a detailed understanding of the process of blood formation. Furthermore, it is also promising for future application in hematopoietic cell and gene therapy. Since the arrival of techniques which allow the reprogramming of somatic cells back to an ES cell-like state, the generation of hematopoietic stem cells from patient-specific so-called induced pluripotent stem cells shows great promise for future therapeutic applications. In this chapter, we describe how to cultivate a certain feeder cell-independent mouse embryonic stem cell line, to manipulate these cells by retroviral gene transfer to ectopically express HOXB4, to differentiate these ES cells via embryoid body formation, and to selectively expand the arising, HOXB4-expressing hematopoietic stem and progenitor cells.

Differentiation of pluripotent embryonic stem (ES) cells can recapitulate many aspects of hematopoiesis, in vitro, and can even generate cells capable of long-term multilineage repopulation after transplantation into recipient mice, when the homeodomain transcription factor HOXB4 is ectopically expressed. Thus, the ES-cell differentiation system is of great value for a detailed understanding of the process of blood formation. Furthermore, it is also promising for future application in hematopoietic cell and gene therapy. Since the arrival of techniques which allow the reprogramming of somatic cells back to an ES cell-like state, the generation of hematopoietic stem cells from patient-specific so-called induced pluripotent stem cells shows great promise for future therapeutic applications. In this chapter, we describe how to cultivate a certain feeder cell-independent mouse embryonic stem cell line, to manipulate these cells by retroviral gene transfer to ectopically express HOXB4, to differentiate these ES cells via embryoid body formation, and to selectively expand the arising, HOXB4-expressing hematopoietic stem and progenitor cells.

Induced pluripotent stem cells (iPSCs) may become a promising source for the generation of patient-specific hematopoietic stem cells (HSCs) in vitro. A crucial prerequisite will be the availability of reliable protocols for the directed and efficient differentiation toward HSCs. So far, the most robust strategy for generating HSCs from pluripotent cells in vitro has been established in the mouse model involving ectopic expression of the human transcription factor HOXB4. However, most differentiation protocols include coculture on a xenogenic stroma cell line and the use of animal serum. Involvement of any of both would pose a major barrier to the translation of those protocols to human autologous iPSCs intended for clinical use. Therefore, we asked whether long-term repopulating HSCs can, in principle, be generated from embryonic stem cells without stroma cells or serum. Here, we showed that long-term multilineage engraftment could be accomplished in immunocompetent mice when HSCs were generated in serum-free medium without stroma cell support and when hypoxic conditions were used. Under those conditions, HOXB4+ embryonic stem cell-derived hematopoietic stem and progenitor cells were immunophenotypically similar to definitive bone marrow resident E-SLAM+ (CD150+CD48−CD45+CD201+) HSCs. Thus, our findings may ease the development of definitive, adult-type HSCs from pluripotent stem cells, entirely in vitro.

Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iPSC, and imaged the conversion of fibroblasts to iPSC. We combined fluorescence microscopy with long-term single cell tracking, and used live-cell imaging to analyze the emergence and composition of early iPSC clusters. Applying our engineered lentiviral vectors, we demonstrate that vector silencing typically occurs prior to or simultaneously with the induction of an Oct4-EGFP pluripotency marker. Around 7 days post-transduction (pt), a subfraction of cells in clonal colonies expressed Oct4-EGFP and rapidly expanded. Cell tracking of single cell-derived iPSC colonies supported the concept that stochastic epigenetic changes are necessary for reprogramming. We also found that iPSC colonies may emerge as a genetic mosaic originating from different clusters. Improved vector design with continuous cell tracking thus creates a powerful system to explore the subtle dynamics of biological processes such as early reprogramming events.

The rate-limiting step of eukaryotic protein synthesis is the binding of mRNA to the 40 S ribosomal subunit, a step which is catalyzed by initiation factors of the eIF-4 (eukaryotic initiation factor 4) group: eIF-4A, eIF-4B, eIF-4E, and eIF-4 gamma. Infection of cells with picornaviruses of the rhino- and enterovirus groups causes a shut-off in translation of cellular mRNAs but permits viral RNA translation to proceed. This change in translational specificity is thought to be mediated by proteolytic cleavage of eIF-4 gamma, which is catalyzed, directly or indirectly, by the picornaviral 2A protease. In this report we have used highly purified recombinant 2A protease from either human Coxsackievirus serotype B4 or rhinovirus serotype 2 to cleave eIF-4 gamma in vitro in the eIF-4 complex purified from rabbit reticulocytes. Neither the rate of cleavage nor fragment sizes were affected by addition of eIF-3. The NH2- and COOH-terminal fragments of eIF-4 gamma were separated by reverse phase HPLC and identified with specific antibodies, and the NH2-terminal sequence of the COOH-terminal fragment was determined by automated Edman degradation. The cleavage site for both proteases is 479GRPALSSR decreases GPPRGGPG494 in rabbit eIF-4 gamma, corresponding to 478GRTTLSTR decreases GPPRGGPG493 in human eIF-4 gamma.

The ability of embryonic stem (ES) cells to form cells and tissues from all 3 germ layers can be exploited to generate cells that can be used to treat diseases. In particular, successful generation of hematopoietic cells from ES cells could provide safer and less immunogenic cells than bone marrow cells, which require severe host preconditioning when transplanted across major histocompatibility complex barriers. Here, we exploited the self-renewal properties of ectopically expressed HOXB4, a homeobox transcription factor, to generate hematopoietic progenitor cells (HPCs) that successfully induce high-level mixed chimerism and long-term engraftment in recipient mice. The HPCs partially restored splenic architecture in Rag2−/−γc−/−–immunodeficient mice. In addition, HPC-derived newly generated T cells were able to mount a peptide-specific response to lymphocytic choriomeningitis virus and specifically secreted interleukin-2 and interferon-γ upon CD3 stimulation. In addition, HPC-derived antigen presenting cells in chimeric mice efficiently presented viral antigen to wild-type T cells. These results demonstrate for the first time that leukocytes derived from ES cells ectopically expressing HOXB4 are immunologically functional, opening up new opportunities for the use of ES cell–derived HPCs in the treatment of hematologic and immunologic diseases.

Homeodomain-containing transcription factors are important regulators of stem cell behavior. HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis with enriched adult HSCs and embryonic derivatives expressing inducible HOXB4. Thereby, we identified a set of overlapping genes that likely represent “universal” targets of HOXB4. A substantial number of loci are involved in signaling pathways important for controlling self-renewal, maintenance, and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-α. This protection likely contributes to the competitive repopulation advantage of HOXB4-expressing HSCs observed in vivo . The concept of TNF-α inhibition may also prove beneficial for patients undergoing bone marrow transplantation. Furthermore, we demonstrate that HOXB4 activity and FGF signaling are intertwined. HOXB4-mediated expansion of adult and ES cell-derived HSCs was enhanced by specific and complete inhibition of FGF receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day 4–6 embryoid bodies was blunted in the presence of basic FGF (FGF2), indicating a dominant negative effect of FGF signaling on the earliest hematopoietic cells. In summary, our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance toward stem cell self-renewal.

Ectopic expression of the homeodomain transcription factor HOXB4 expands hematopoietic stem and progenitor cells in vivo and in vitro, making HOXB4 a highly interesting candidate for therapeutic stem cell expansion. However, when expressed at high levels, HOXB4 concomitantly perturbs differentiation and thus likely predisposes the manipulated cells for leukemogenesis. We therefore asked whether the expression level of HOXB4 may be a critical parameter that influences the growth and transformation properties of transduced cells. Using a set of retroviral vectors which covered a 40-fold range of expression levels, we studied the consequences of HOXB4 expression at different levels in the well established Rat-1 fibroblast cell system. HOXB4 transformed Rat-1 fibroblasts beyond a certain threshold level of expression. Further escalation of HOXB4 expression, however, did not enhance transformation. Instead, HOXB4 mediated a dose dependent anti-proliferative effect on Rat-1 and NIH3T3 fibroblasts. This effect was aggravated under reduced serum concentrations and was, at least partially, due to an enhanced sensitivity of HOXB4 overexpressing cells to induction of apoptosis. Based on these results we propose that HOXB4 affects cell growth in a dose-dependent manner by sensitizing cells towards extrinsic signals.

Three RNA features have been identified that elevate retroviral transgene expression: an intron in the 5' untranslated region (5'UTR), the absence of aberrant translational start codons and the presence of the post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus in the 3'UTR. To include such elements into self-inactivating (SIN) vectors with potentially improved safety, we excised the strong retroviral promoter from the U3 region of the 3' long terminal repeat (LTR) and inserted it either downstream or upstream of the retroviral RNA packaging signal (Psi). The latter concept is new and allows the use of an intron in the 5'UTR, taking advantage of retroviral splice sites surrounding Psi. Three LTR and four SIN vectors were compared to address the impact of RNA elements on titer, splice regulation and transgene expression. Although titers of SIN vectors were about 20-fold lower than those of their LTR counterparts, inclusion of the PRE allowed production of more than 10(6) infectious units per ml without further vector optimizations. In comparison with state-of-the-art LTR vectors, the intron-containing SIN vectors showed greatly improved splicing. With regard to transgene expression, the intron-containing SIN vectors largely matched or even exceeded the LTR counterparts in all cell types investigated (embryonic carcinoma cells, fibroblasts, primary T cells and hematopoietic progenitor cells).

The aim of this study was to determine the impact of lentiviral transduction on primary murine B cells. Studying B cell activities in vivo or using them for tolerance induction requires that the cells remain unaltered in their biological behavior except for expression of the transgene. As we show here, murine B cells can efficiently be transduced by lentiviral, VSV-G-pseudotyped vectors without the necessity of prior activation. Culture with LPS gave enhanced transduction efficiencies but led to the upregulation of CD86 and proliferation of the cells. Transduction of naive B cells by lentiviral vectors was dependent on multiplicity of infection and did not lead to a concomitant activation. Furthermore, the transduced cells could be used for studies in the NOD mouse system without altering the onset of diabetes. We conclude that lentiviral gene transfer into naive B cells is a powerful tool for manipulation of B cells for therapeutic applications.

Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for future somatic gene and cell therapy. So far, HSCs capable of long-term multilineage reconstitution in mice have only been obtained when the homeodomain transcription factor HOXB4 was ectopically expressed during pluripotent stem cell differentiation (Kyba et al. Cell 109(1): 29-37, 2002; Pilat et al. Proc Natl Acad Sci USA 102(34): 12101-12106, 2005; Lesinski et al. Stem Cells Transl Med 1(8): 581-591, 2012). However, the primary "target" cell of HOXB4 during hematopoietic development, in vitro, is not yet known. Its identification is a prerequisite for unambiguously identifying the molecular circuits driving HSC development, at least in vitro. To pin down this cell, we retrovirally expressed HOXB4 or a Tamoxifen-inducible HOXB4-ERT2 fusion protein in different reporter and knock-out mouse embryonic stem cell (ESC) lines. For these experiments, ESCs were differentiated for 6 days as embryoid bodies (EBs), dissociated and subsequently cocultured on OP9 stroma cells in medium supplemented with 100 ng/ml mSCF, 40 ng/ml mTPO, 100 ng/ml hFlt3L and 40 ng/ml hVEGF (STFV) for further 3 days. Use of a Runx1(-/-) ESC-line containing a doxycycline-inducible Runx1 coding sequence (“iRunx1”; kindly provided by G. Lacaud, Manchester) uncovered that HOXB4 acts during formation of the hemogenic endothelium (HE) from which HSCs arise. Without Runx1 induction, which arrests hematopoietic development at the HE-stage, ectopic HOXB4 expression mediated an approximately 30-fold increase in the number of circular endothelial, bona fide HE-sheets being Flk1+VE-Cadherin+Tie2+ (mean values: control: 11+/-4.8 n=7; HOXB4: 301+/-47 n=7; P Taken together, our results strongly suggest that HOXB4 first and foremost promotes hematopoiesis by substantially increasing the number of hemogenic endothelium progenitors during mouse pluripotent stem cell differentiation. Disclosures No relevant conflicts of interest to declare.

This study presents a promising selection system for gene-modified cells other than human hematopoietic progenitor and endothelial cells based on transgenic expression of human CD34. Three retrovirally transduced variants of CD34 were compared, differing in the length of their cytoplasmic domains. These were the full-length transmembrane protein (flCD34), a truncated form (tCD34) that is found as a naturally occurring splice variant and has a partial deletion of the cytoplasmic domain for signal transduction, and an engineered variant which is completely deprived of its cytoplasmic tail (dCD34). All three variants allowed selection of gene-modified cells using commercially available immunoaffinity technology. However, examination by flow cytometry as well as by Southern, Northern, and Western blot revealed that dCD34, as opposed to tCD34, is not stably anchored in the membrane and thus is expressed at low levels on the surface of transduced cells. Therefore, tCD34 was chosen as the more promising candidate for a clinically applicable cell surface marker. We show that gene-modified human primary T lymphocytes expressing tCD34 can be enriched to high purity (>95%) using clinically approved immunoaffinity columns. In addition, we demonstrate the utility of tCD34 for surface marking of murine hematopoietic cells in vivo, including primary T lymphocytes detected 9 weeks after bone marrow transplantation.

Retroviral vector-mediated expression of the homeoboxgene, HoxB4, in hematopoietic cells has been reported to mediate a benign expansion of gene-modified hematopoietic stem and precursor cells in vivo. In the present study, we used a novel coexpression strategy for coordinated expression of HoxB4 along with a cytoplasmic protein from a retroviral vector. The novel coexpression strategy, based on cotranslational protein separation mediated by the 2A sequence of foot-and-mouth disease virus (FMDV), allows an indirect quantification of HoxB4 expression levels when inserting a reporter such as the enhanced green fluorescent protein (GFP) in the retroviral vector. Presence of the 2A sequence did not interfere with the correct subcellular localization of HoxB4 (nuclear) and GFP (cytoplasmic), nor with the titer of bicistronic vectors, and mediated functional long-term coexpression (at least 1 year) of GFP and HoxB4 after transplantation of transduced mouse bone marrow cells in mice.