Cell Origin of Human Mesenchymal Stem Cells Determines a Different Healing Performance in Cardiac Regeneration

The possible different therapeutic efficacy of human mesenchymal stem cells (hMSC) derived from umbilical cord blood (CB), adipose tissue (AT) or bone marrow (BM) for the treatment of myocardial infarction (MI) remains unexplored. This study was to assess the regenerative potential of hMSC from different origins and to evaluate the role of CD105 in cardiac regeneration. Male SCID mice underwent LAD-ligation and received the respective cell type (400.000/per animal) intramyocardially. Six weeks post infarction, cardiac catheterization showed significant preservation of left ventricular functions in BM and CD105+-CB treated groups compared to CB and nontreated MI group (MI-C). Cell survival analyzed by quantitative real time PCR for human GAPDH and capillary density measured by immunostaining showed consistent results. Furthermore, cardiac remodeling can be significantly attenuated by BM-hMSC compared to MI-C. Under hypoxic conditions in vitro, remarkably increased extracellular acidification and apoptosis has been detected from CB-hMSC compared to BM and CD105 purified CB-derived hMSC. Our findings suggests that hMSC originating from different sources showed a different healing performance in cardiac regeneration and CD105+ hMSC exhibited a favorable survival pattern in infarcted hearts, which translates into a more robust preservation of cardiac function

Kryokonservierung von humanen vaskulären Nabelschnurzellen unter Bedingungen angepasst an die Regularien der "Guten Herstellungspraxis" für künftige Zellbanken

Introduction: Cardiovascular disease is the leading cause of death and disability worldwide and estimated 23.6 million people will die from the disease in 2030. In vitro fabricated tissue engineered vascular constructs could provide an alternative to conventional substitutes such as mechanical and biological grafts which are currently satisfactorily used as implants in cardiac surgery. Vascular cells from the human umbilical cord represent an appropriate cell source because they are similar to the native cellular structures and functionalities found in heart valves and they can be directly isolated and cryopreserved until needed. Use of these cells for therapeutic applications such as tissue engineered cardiovascular constructs requires the establishment of a human vascular cell bank conforming to Good Manufacturing Practice (GMP) conditions. Methodology: The replacement of conventional research and development starting materials by GMP compliant starting materials was a crucial step during isolation, cultivation and cryopreservation process of human umbilical cord artery derived cells (HUCAC) and human umbilical vein endothelial cells (HUVEC). To analyse the influence of cryopreservation, the cells were short and long term cyropreserved directly after primary culture, and recultivated subsequently. Fresh and cryopreserved cells were studied concerning cell viability, expression of cellular markers and proliferation potential using trypan blue staining, flow cytometry analysis, immunofluorescence staining and proliferation assays. Results: Cell isolation was successful using GMP compliant starting materials since sufficient numbers of cells with acceptable viabilities and homogenous expression of cellular markers were obtained. Cryopreserved cells mostly maintain phenotypic and functional characteristics similarly to fresh cells, confirming that the influence of cryopreservation was negligible. Consequently, fresh cultivated and cryopreserved HUCAC expressed alpha smooth muscle actin, CD90, CD105, CD73, CD29, CD44, CD166 but not smoothelin, and HUVEC were positive for CD31, CD146, CD105 and CD144 but negative for alpha smooth muscle actin. Acceptable viability and sufficient proliferation properties of cryopreserved HUCAC and HUVEC were analysed in functional studies. Conclusion: Cell isolation, cultivation and cryopreservation were successfully adapted to GMP compliant starting materials. The influence of cryopreservation to cell properties was marginal and without lasting effect confirming that vascular cells from the human umbilical cord represent a potential cell source for cell banking. Using flow cytometry analysis a specific cellular marker expression profile was developed for HUCAC and HUVEC, usable as a GMP compliant quality control. For the future fabrication of advanced therapy medicinal products consisting of these cells, GMP conditions are required by the regulatory authority.Einleitung: Kardiovaskuläre Erkrankungen stellen weltweit die häufigste Ursache für Todesfälle und Behinderungen dar. Bis 2030 werden voraussichtlich 23,6 Millionen Menschen an diesen Erkrankungen sterben. Die in vitro Herstellung von kardiovaskulären Substituten mittels Tissue Engineering (TE) könnte eine mögliche Alternative zu den derzeit etablierten mechanischen und biologischen Ersatzprothesen für Gefäße und Herzklappen bieten. Vaskuläre Zellen der humanen Nabelschnur könnten sich für das TE als Zellquelle eignen, da diese den nativen Zellen der Herzklappe in Struktur und Funktionalität ähneln. Sie können direkt isoliert und als individuelle bzw. allogene Zellquelle kryokonserviert werden. Die Verwendung dieser Zellen für therapeutische Zwecke, wie zum Beispiel das TE kardiovaskulärer Substitute, erfordert die Entwicklung einer humanen vaskulären Zellbank, die an die Bedingungen der Good Manufacturing Practice (GMP) angepasst ist. Methodik: Einen wesentlichen Meilenstein stellte eine konsequente Adaption der gängigen Forschungs- und Entwicklungs- Reagenzien an pharmazeutisch einsetzbare, GMP konforme Ausgangsstoffe während der Isolierung, Kultivierung und Kryokonservierung von human umbilical cord artery derived cells (HUCAC) und human umbilical vein endothelial cells (HUVEC) dar. Um den Einfluss der Kryokonservierung auf die Zellen zu untersuchen, wurden diese direkt nach der Primärkultur kurzzeit- und langzeitkryokonserviert und anschließend rekultiviert. Zellvitalität, Expression zellulärer Marker und Proliferationspotential frisch kultivierter und kryokonservierter Zellen wurden unter Anwendung der Trypanblau-Färbung, Durchflusszytometrie, Immunfluoreszenzfärbung und des Proliferationsassay untersucht. Ergebnisse: Unter Verwendung von GMP konformen Ausgangsstoffen wurde eine zufrieden stellende Anzahl von Zellen aus den Gefäßwänden der Nabelschnur isoliert und die Zellen wiesen eine akzeptable Vitalität sowie eine homogene Expression zellulärer Marker auf. Der Einfluss der Kryokonservierung auf die Zellen erwies sich als geringfügig denn die phänotypischen und funktionellen Eigenschaften waren weitgehend mit denen frisch kultivierter Zellen vergleichbar. Frisch kultivierte und kryokonservierte HUCAC exprimierten alpha smooth muscle actin (ASMA), CD90, CD105, CD73, CD29, CD44, CD166, jedoch nicht smoothelin. HUVEC zeigten positive Signale für CD31, CD146, CD105 und CD144, ASMA konnte hingegen nicht detektiert werden. In funktionellen Analysen konnten eine akzeptable Vitalität und ein zufrieden stellendes Wachstumsverhalten der HUCAC und HUVEC ermittelt werden. Schlussfolgerung: Die Zellisolierung, Kultivierung und Kryokonservierung wurde erfolgreich an GMP konforme Ausgangsstoffe angepasst. Die Kryokonservierung beeinflusste die Eigenschaften der Zellen nur geringfügig und ohne nachhaltige Auswirkungen, weshalb die Verwendung dieser vaskulären Nabelschnurzellen für eine Zellbank durchaus möglich ist. Ein zellspezifisches Markerprofil wurde für die HUCAC und HUVEC mit Hilfe der Duchflusszytometrie etabliert, welches als GMP konforme Qualitätskontrolle einsetzbar wäre. Für die zukünftige Herstellung von Medizinprodukten aus diesen Zellen wird von den Kontrollbehörden eine GMP- Konformität für alle Herstellungsstufen gefordert

За кадры. 1976. № 73 (1991)

Счастливая судьба / П. ТушинПервые патенты химиков / В. БогатыревНужны наставники. Интервью на актуальную тему / [беседа с] С. С. Сулакшин ; [беседа с] П. В. РожковМгновения, мгновения, мгновения... Скоро сессияГлавный стержень. Качество нашей работы / Р. ГорскаяЕе дело / Л. РезановаНеважные дела гаражные. С открытого партийного собрания АХУ / А. МоскалевБорьба без снисхождения. Пьянству - бой / О. НиколаеваИсключены и отчислены / П. Богданов, В. СпиридоновПрекрасная постановка / В. СоколовПустуют Красные уголки / В. РябковВклад политехников в тракторостроение / И. Т. Лозовски

Real time acidification rate as a live cell parameter.

<p>CB derived hMSC show significant increased metabolic activity under hypoxic situation compared to BM- and CD105-purified CB-derived hMSC.</p

CD105 cell isolated from BM hMSC were transiently transfected with GFP, antisense-Oligodesoxynukleotide (ODN-CD105), scramble ODN.

<p><b><i>A.</i></b> Transfection efficiency is shown on the upper picture (GFP) <b><i>B.</i></b> Silencing of CD105 blocks BM-hMSC tube formation compared to nature and scrambled group. The BM-hMSC were embedded in Matrigel and incubated in EGM-2 seven days after transfection and then imaged. <i>C.</i> Cell proliferation was analyzed at 24, 48 and 96 h after the ODN-CD105 and scramble ODN transfection by MTT assay.</p

CD105 expression in CB105⁺ hMSC over the culture time.

<p>n = 3; mean [%]±SEM.</p

Heart functions 6 weeks after MI.

<p><b><i>A.</i></b> Recovery of cardiac performance shows improvement for hearts with implanted human BM- and CD105-purified CB-hMSC compared to MI-CB hearts. <b><i>B.</i></b> Left ventricular functions at both baseline and stress condition assessed by catheterization.</p

Identification of transplanted hMSC in infracted myocardium.

<p>6 weeks after MI: <b><i>A–C.</i></b> Representative immunofluorescent micrographs of hearts transplanted with hMSC. <b><i>A</i></b> transplanted hMSC could be identified in infarcted myocardium <b><i>B.</i></b> A number of hMSC (Arrows, human nuclei in green) were co-localized with CD31 positive cells (red). <b><i>C.</i></b> Occasionally hMSC (Arrow, human nuclei in green) co-localized with cardiac troponin positive cell (red). (Confocal image, original magnification 630×) <b><i>D.</i></b> Quantitative real-time PCR analysis for human GAPDH expression level at different infarction sections: MI-BM and MI-CB105⁺ hearts show significantly higher localisation of human cells in the middle and apex section.</p

Fibrosis 6 weeks after MI.

<p><b><i>A.</i></b> Representative Fast Green FCF (myocytes)/Sirius Red (fibrosis) stainings at the BZ. <b><i>B.</i></b> Significantly decrease of collagen deposition has been shown in both the RA and the BZ in MI-CB105⁺ compared to MI-CB and MI-C.</p

Phenotypic characterization of hMSC from different sources.

<p><b><i>A.</i></b> FACS analysis showed that the cells were negative for CD45 expression and positive for CD29, CD44, CD73 and CD105, which are phenotypes currently known to be characteristic of hMSC. The gray line indicates the control of the CD marker isotypes. <b><i>B.</i></b><i> In vitro</i> differentiation capacity of transplanted hMSC. hMSC from bone marrow were cultured in adipogenic and chondrogenic medium. Chondrogenic differentiation (left). Immunostaining for aggrecan (red). Nuclei were counterstained with DAPI (blue). Adipogenic differentiation (right). Immunostaining with fatty acid binding protein-4 (brown).</p