Transcriptome Analysis Reveals High Similarities between Adult Human Cardiac Stem Cells and Neural Crest-Derived Stem Cells

For the identification of a stem cell population, the comparison of transcriptome data enables the simultaneous analysis of tens of thousands of molecular markers and thus enables the precise distinction of even closely related populations. Here, we utilized global gene expression profiling to compare two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells (ITSCs) of the nasal cavity and human cardiac stem cells (hCSCs) from the heart auricle. We detected high similarities between the transcriptomes of both stem cell populations, particularly including a range of neural crest-associated genes. However, global gene expression likewise reflected differences between the stem cell populations with regard to their niches of origin. In a broader analysis, we further identified clear similarities between ITSCs, hCSCs and other adherent stem cell populations compared to non-adherent hematopoietic progenitor cells. In summary, our observations reveal high similarities between adult human cardiac stem cells and neural crest-derived stem cells from the nasal cavity, which include a shared relation to the neural crest. The analyses provided here may help to understand underlying molecular regulators determining differences between adult human stem cell populations

Blood plasma-mediated effects on regenerative features of a novel adult human cardiac stem cell population

Höving A. Blood plasma-mediated effects on regenerative features of a novel adult human cardiac stem cell population. Bielefeld: Universität Bielefeld; 2021.During lifetime, the human organism is exposed to a progressive decline in organ function and regeneration considered as the ageing process. Several age-associated diseases such as cardiovascular- and neurodegenerative diseases or cancer are described along with their cellular pathologies. Although tissue regeneration substantially relies on the functionality of endogenous adult stem cell populations found in various organs including the heart, a complex interaction with systemic factors carried by the blood is evident. Thus, young blood or blood products are increasingly recognized as potential therapeutic agents to target age-associated malignancies. In this regard, prominent studies in the murine model gained attention by rejuvenating old mice with blood or blood products from young mice. However, the transition of these promising results to the human system remains challenging. Here, adequate cellular models need to be established to study the effects of human blood plasma on the regeneration of human tissues and particularly the heart. Facing this challenge, this thesis describes the age-specific effects of human blood plasma and blood serum on a human cardiac stem cell (hCSC)-based model for heart regeneration in terms of proliferation, migration, senescence, and global gene expression. Here, the identification of a novel multipotent hCSC population from the heart auricle of patients undergoing routine cardiac surgery enabled the establishment of a cellular model to study human cardiac regeneration. These cells can easily be isolated and expanded in culture and express common markers for cardiac stem cell populations. Moreover, hCSCs showed neural crest-specific stem cell markers and high transcriptional similarities with a known neural crest-derived stem cell population. Their differentiation capacities into mesodermal as well as neuro-ectodermal derivates further suggest a potential relation to the neural crest. Treatment of hCSCs with human plasma and serum greatly induced their proliferation with no significant differences regarding age and sex of the plasma donors. In contrast, age-dependent effects were detectable in the serum-mediated protection against senescence with serum from old (> 60 years) female donors showing the highest rate of protection compared to male or young female donors. Further, the migrative capacities of hCSCs in terms of migration distance and velocity were significantly increased after serum treatment. A global transcriptomic analysis of serum-treated hCSCs revealed an age-dependent increase of differential gene expression in hCSCs treated with young sera compared to untreated cells and the upregulation of genes associated to the p38 mitogen-activated protein kinase (p38-MAPK)-pathway. Pharmacological inhibition of p38-MAPK significantly reduced the beneficial effects of human blood serum in terms of decreased proliferation and migration as well as increased senescence. Next to its beneficial effects on the successfully established cellular model system for cardiac ageing and regeneration, human serum was applied to *ex vivo* cultured mouse hippocampal slices as an experimental model for neurodegenerative diseases. Here, human blood plasma as well as human serum albumin (HSA) as the most abundant plasma protein revealed significant neuroprotective effects against kainic acid (KA)-mediated neuronal cell death compared to untreated hippocampal slice cultures. In summary, the here presented results show the identification of a novel hCSC population and its developmental relation to the neural crest as well as its successful application as a screening system for human blood plasma-mediated regenerative responses. On functional level, p38-MAPK was identified as crucial mediator of the blood-plasma-based induction of hCSC-proliferation and -migration as well as protection of hCSCs against senescence. Future studies will carefully investigate the regulatory mechanisms upstream and downstream of p38-MAPK signaling in serum-treated hCSCs and might enable the identification of the responsible plasma components. Finally, this work provides valuable insights into the beneficial effects of human blood plasma on the regenerative function of adult human cardiac stem cells and builds a basis for the potential clinical use of human blood plasma as well as cardiac stem cells in regenerative medicine

Transcriptome Analysis Reveals High Similarities between Adult Human Cardiac Stem Cells and Neural Crest-Derived Stem Cells

Höving AL, Sielemann K, Greiner J, Kaltschmidt B, Knabbe C, Kaltschmidt C. Transcriptome Analysis Reveals High Similarities between Adult Human Cardiac Stem Cells and Neural Crest-Derived Stem Cells. Biology. 2020;9(12): 435.For the identification of a stem cell population, the comparison of transcriptome data enables the simultaneous analysis of tens of thousands of molecular markers and thus enables the precise distinction of even closely related populations. Here, we utilized global gene expression profiling to compare two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells (ITSCs) of the nasal cavity and human cardiac stem cells (hCSCs) from the heart auricle. We detected high similarities between the transcriptomes of both stem cell populations, particularly including a range of neural crest-associated genes. However, global gene expression likewise reflected differences between the stem cell populations with regard to their niches of origin. In a broader analysis, we further identified clear similarities between ITSCs, hCSCs and other adherent stem cell populations compared to non-adherent hematopoietic progenitor cells. In summary, our observations reveal high similarities between adult human cardiac stem cells and neural crest-derived stem cells from the nasal cavity, which include a shared relation to the neural crest. The analyses provided here may help to understand underlying molecular regulators determining differences between adult human stem cell populations

Identification of a Novel High Yielding Source of Multipotent Adult Human Neural Crest-Derived Stem Cells

Schürmann M, Brotzmann V, Bütow M, et al. Identification of a Novel High Yielding Source of Multipotent Adult Human Neural Crest-Derived Stem Cells. Stem Cell Reviews and Reports. 2018;14(2):277–285.Due to their extraordinarily broad differentiation potential and persistence during adulthood, adult neural crest-derived stem cells (NCSCs) are highly promising candidates for clinical applications, particularly when facing the challenging treatment of neurodegenerative diseases or complex craniofacial injuries. Successful application of human NCSCs in regenerative medicine and pharmaceutical research mainly relies on the availability of sufficient amounts of tissue for cell isolation procedures. Facing this challenge, we here describe for the first time a novel population of NCSCs within the middle turbinate of the human nasal cavity. From a surgical point of view, high amounts of tissue are routinely and easily removed during nasal biopsies. Investigating the presence of putative stem cells in obtained middle turbinate tissue by immunohistochemistry, we observed Nestin+/p75NTR+/S100+/α smooth muscle actin (αSMA)− cells, which we successfully isolated and cultivated in vitro. Cultivated middle turbinate stem cells (MTSCs) kept their expression of neural crest and stemness markers Nestin, p75 and S100 and showed the capability of sphere formation and clonal growth, indicating their stem cell character. Application of directed in vitro differentiation assays resulted in successful differentiation of MTSCs into osteogenic and neuronal cell types. Regarding the high amount of tissue obtained during surgery as well as their broad differentiation capability, MTSCs seem to be a highly promising novel neural crest stem cell population for applications in cell replacement therapy and pharmacological research

Between Fate Choice and Self-Renewal - Heterogeneity of Adult Neural Crest-Derived Stem Cells

Höving AL, Windmöller BA, Knabbe C, Kaltschmidt B, Kaltschmidt C, Greiner J. Between Fate Choice and Self-Renewal - Heterogeneity of Adult Neural Crest-Derived Stem Cells. Frontiers in Cell and Developmental Biology. 2021;9: 662754.Stem cells of the neural crest (NC) vitally participate to embryonic development, but also remain in distinct niches as quiescent neural crest-derived stem cell (NCSC) pools into adulthood. Although NCSC-populations share a high capacity for self-renewal and differentiation resulting in promising preclinical applications within the last two decades, inter- and intrapopulational differences exist in terms of their expression signatures and regenerative capability. Differentiation and self-renewal of stem cells in developmental and regenerative contexts are partially regulated by the niche or culture condition and further influenced by single cell decision processes, making cell-to-cell variation and heterogeneity critical for understanding adult stem cell populations. The present review summarizes current knowledge of the cellular heterogeneity within NCSC-populations located in distinct craniofacial and trunk niches including the nasal cavity, olfactory bulb, oral tissues or skin. We shed light on the impact of intrapopulational heterogeneity on fate specifications and plasticity of NCSCs in their nichesin vivoas well as duringin vitroculture. We further discuss underlying molecular regulators determining fate specifications of NCSCs, suggesting a regulatory network including NF-κB and NC-related transcription factors like SLUG and SOX9 accompanied by Wnt- and MAPK-signaling to orchestrate NCSC stemness and differentiation. In summary, adult NCSCs show a broad heterogeneity on the level of the donor and the donors’ sex, the cell population and the single stem cell directly impacting their differentiation capability and fate choicesin vivoandin vitro. The findings discussed here emphasize heterogeneity of NCSCs as a crucial parameter for understanding their role in tissue homeostasis and regeneration and for improving their applicability in regenerative medicine

Serum-Induced Proliferation of Human Cardiac Stem Cells Is Modulated via TGFβRI/II and SMAD2/3

Schmidt KE, Höving AL, Kiani Zahrani S, et al. Serum-Induced Proliferation of Human Cardiac Stem Cells Is Modulated via TGFβRI/II and SMAD2/3. International Journal of Molecular Sciences. 2024;25(2): 959.The ageing phenotype is strongly driven by the exhaustion of adult stem cells (ASCs) and the accumulation of senescent cells. Cardiovascular diseases (CVDs) and heart failure (HF) are strongly linked to the ageing phenotype and are the leading cause of death. As the human heart is considered as an organ with low regenerative capacity, treatments targeting the rejuvenation of human cardiac stem cells (hCSCs) are of great interest. In this study, the beneficial effects of human blood serum on proliferation and senescence of hCSCs have been investigated at the molecular level. We show the induction of a proliferation-related gene expression response by human blood serum at the mRNA level. The concurrent differential expression of the TGFβ target and inhibitor genes indicates the participation of TGFβ signalling in this context. Surprisingly, the application of TGFβ1 as well as the inhibition of TGFβ type I and type II receptor (TGFβRI/II) signalling strongly increased the proliferation of hCSCs. Likewise, both human blood serum and TGFβ1 reduced the senescence in hCSCs. The protective effect of serum on senescence in hCSCs was enhanced by simultaneous TGFβRI/II inhibition. These results strongly indicate a dual role of TGFβ signalling in terms of the serum-mediated effects on hCSCs. Further analysis via RNA sequencing (RNA-Seq) revealed the participation of Ras-inactivating genes wherefore a prevention of hyperproliferation upon serum-treatment in hCSCs via TGFβ signalling and Ras-induced senescence is suggested. These insights may improve treatments of heart failure in the future

Transcriptome analysis reveals high similarities between adult human cardiac stem cells and neural crest-derived stem cells

For the identification of a stem cell population, the comparison of transcriptome data enables the simultaneous analysis of tens of thousands of molecular markers and thus enables the precise distinction of even closely related populations. Here, we utilized global gene expression profiling to compare two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells (ITSCs) of the nasal cavity and human cardiac stem cells (hCSCs) from the heart auricle. We detected high similarities between the transcriptomes of both stem cell populations, particularly including a range of neural crest-associated genes. However, global gene expression likewise reflected differences between the stem cell populations with regard to their niches of origin. In a broader analysis, we further identified clear similarities between ITSCs, hCSCs and other adherent stem cell populations compared to non-adherent hematopoietic progenitor cells. In summary, our observations reveal high similarities between adult human cardiac stem cells and neural crest-derived stem cells from the nasal cavity, which include a shared relation to the neural crest. The analyses provided here may help to understand underlying molecular regulators determining differences between adult human stem cell populations

Human Blood Serum Induces p38-MAPK- and Hsp27-Dependent Migration Dynamics of Adult Human Cardiac Stem Cells: Single-Cell Analysis via a Microfluidic-Based Cultivation Platform

Schmitz J, Höving AL, Schmidt KE, et al. Human Blood Serum Induces p38-MAPK- and Hsp27-Dependent Migration Dynamics of Adult Human Cardiac Stem Cells: Single-Cell Analysis via a Microfluidic-Based Cultivation Platform. Biology. 2021;10(8): 708.Migratory capabilities of adult human stem cells are vital for assuring endogenous tissue regeneration and stem cell-based clinical applications. Although human blood serum has been shown to be beneficial for cell migration and proliferation, little is known about its impact on the migratory behavior of cardiac stem cells and underlying signaling pathways. Within this study, we investigated the effects of human blood serum on primary human cardiac stem cells (hCSCs) from the adult heart auricle. On a technical level, we took advantage of a microfluidic cultivation platform, which allowed us to characterize cell morphologies and track migration of single hCSCs via live cell imaging over a period of up to 48 h. Our findings showed a significantly increased migration distance and speed of hCSCs after treatment with human serum compared to control. Exposure of blood serum-stimulated hCSCs to the p38 mitogen-activated protein kinase (p38-MAPK) inhibitor SB239063 resulted in significantly decreased migration. Moreover, we revealed increased phosphorylation of heat shock protein 27 (Hsp27) upon serum treatment, which was diminished by p38-MAPK-inhibition. In summary, we demonstrate human blood serum as a strong inducer of adult human cardiac stem cell migration dependent on p38-MAPK/Hsp27-signalling. Our findings further emphasize the great potential of microfluidic cultivation devices for assessing spatio-temporal migration dynamics of adult human stem cells on a single-cell level

Neuroprotection Mediated by Human Blood Plasma in Mouse Hippocampal Slice Cultures and in Oxidatively Stressed Human Neurons

Ruiz-Perera LM, Höving A, Schmidt K, et al. Neuroprotection Mediated by Human Blood Plasma in Mouse Hippocampal Slice Cultures and in Oxidatively Stressed Human Neurons. International Journal of Molecular Sciences. 2021;22(17): 9567.Neuroprotection from oxidative stress is critical during neuronal development and maintenance but also plays a major role in the pathogenesis and potential treatment of various neurological disorders and neurodegenerative diseases. Emerging evidence in the murine system suggests neuroprotective effects of blood plasma on the aged or diseased brain. However, little is known about plasma-mediated effects on human neurons. In the present study, we demonstrate the neuroprotective effect mediated by human plasma and the most abundant plasma–protein human serum albumin against oxidative stress in glutamatergic neurons differentiated from human neural crest-derived inferior turbinate stem cells. We observed a strong neuroprotective effect of human plasma and human serum albumin against oxidative stress-induced neuronal death on the single cell level, similar to the one mediated by tumor necrosis factor alpha. Moreover, we detected neuroprotection of plasma and human serum albumin against kainic acid-induced excitatory stress in ex vivo cultured mouse hippocampal tissue slices. The present study provides deeper insights into plasma-mediated neuroprotection ultimately resulting in the development of novel therapies for a variety of neurological and, in particular, neurodegenerative diseases

Ultra-fast one-step RT-PCR protocol for the detection of SARS-CoV-2

Asghari E, Höving A, van Heijningen P, et al. Ultra-fast one-step RT-PCR protocol for the detection of SARS-CoV-2. medRxiv. 2020.The COVID-19 pandemic resulted in lockdowns all over the world thus affecting nearly all aspects of social life and also had a huge impact on global economies. Since vaccines and therapies are still not available for the population, prevention becomes desperately needed. One important aspect for prevention is the identification and subsequent isolation of contagious specimens. The currently used methods for diagnostics are time consuming and also hindered by the limited availability of reagents and reaction costs, thus presenting a bottle neck for prevention of COVID-19 spread. Here, we present a new ultra-fast test method which is ten times faster than conventional diagnostic tests using real time quantitative PCR (RT-qPCR). In addition, this ultra-fast method is easy to handle as well as cost effective. We translated published SARS-CoV-2 testing protocols from the Centers of Disease Control and Prevention (Atlanta, Georgia, USA) and the Charité Berlin (Germany) to the NEXTGENPCR (NGPCR) machine and combined it with a fluorescence-based endpoint measurement. Fluorescence was measured with a commercial blue light scanner. We confirmed the NEXTGENPCR results with commercially available positive controls. In addition, we isolated RNA from SARS-CoV-2 infected patients and achieved similar results to clinical RT-qPCR assays. Here, we could show correlation between the results obtained by NEXTGENPCR and conventional RT-qPCR