Extra-hematopoietic immunomodulatory role of the SCID-susceptibility gene DOCK-2 identified by stepwise maturation of human iPSCs into clonogenic mesodermal stromal progenitors

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Stromal cells contribute to organ integrity as fibroblasts and to vascular stability as pericytes, in addition to their enigmatic niche function in many tissues. Their inherent immunomodulatory capacity attracted particular attention, initiating numerous clinical trials, particularly testing trophic regeneration and immunomodulation. Key stromal immune functions are still enigmatic.</jats:p><jats:p>Here we show that dedicator of cytokinesis (DOCK-2) previously described for causing immune cell dysfunction plays a role in extra-hematopoietic immunity by regulating stromal/fibroblast immunomodulatory function. We used three independent strategies including iPSC-derived mesodermal stromal cell (MSC) lineage maturation, severe combined immunodeficiency (SCID) patient-derived cells and CRISPR/Cas9 knockout to support our findings.</jats:p><jats:p>Human induced pluripotent stem cells (iPSCs) were generated from healthy bone marrow and umbilical cord blood-derived fibroblasts by Sendai virus-mediated transient expression of Yamanaka factors. After mesoderm induction, stromal differentiation was induced by platelet-derived growth factors under animal serum-free conditions. Under feeder-free defined conditions, iPSCs differentiated into expandable and cryo-preservable CD73<jats:sup>+</jats:sup>/CD105<jats:sup>+</jats:sup>/Tra-1-81<jats:sup>−</jats:sup> early iPS-MSCs lacking immunosuppressive potential. Successive maturation was required for reaching the canonical MSC phenotype and immunomodulatory competence over time, while maintaining clonogenicity, comparable to parental MSCs. Sequential RNAseq revealed acquisition of a spectrum of immune-related genes significantly expressed in mature iPS-MSCs and resembling parental MSC’s immune gene expression. The DOCK-2 gene attracted our attention because mutations can cause SCID. Interestingly, SCID patient-derived fibroblast lines harboring bi-allelic DOCK-2 mutations showed significantly reduced immunomodulatory capacity compared to non-mutated control fibroblasts. CRISPR/Cas9-mediated DOCK-2 knockout in healthy iPSCs resulted in iPS-MSCs that also displayed reduced immunomodulatory capacity, thus confirming a role of DOCK-2 in stromal immune function. At a mechanistic level, DOCK-2 deficiency resulted in disturbed subcellular localization of CDC42.</jats:p><jats:p>This provides first evidence for an extra-hematopoietic immunomodulatory role of DOCK-2 in stromal cells, previously considered restricted to hampering immune cell migration/function resulting in SCID. We may speculate that some of the signs and symptoms of persisting immune disease after successful hematopoietic stem cell transplantation in SCID patients could at least in part be due to mutations, like DOCK-2<jats:sup>−/−</jats:sup>, permissive outside the hematopoietic immune system, as evidenced also by the increased virus infection susceptibility of DOCK-2 deficient fibroblasts.</jats:p>

Hypoxic Conditions Promote the Angiogenic Potential of Human Induced Pluripotent Stem Cell-Derived Extracellular Vesicles

“…Human stromal cell samples were collected from healthy volunteers after written informed consent according to the Declaration of Helsinki. For genetic reprogramming, using a non-integrative Sendai viral vector kit (CytoTune™-iPS Sendai Reprogramming Kit encoding for Oct4, Sox2, KLF4 and c-Myc, Life Technologies, Carlsbad, CA, USA) was used and performed at the Harvard Stem Cell Institute (HSCI) iPSC Core Facility (Cambridge, MA, USA) adapted from an established protocol as previously described [ 7 , 46 ]. Human iPSCs were cultured in six-well plates (Corning, Corning, NY, USA) coated with Matrigel ® (Corning, USA) in mTeSR™1 medium (STEMCELL Technologies, Vancouver, CB, Canada).…”

Self-assembly of progenitor cells under the aegis of platelet factors facilitates human skin organoid formation and vascularized wound healing

“…For dermal hiPSC-FB generation, mesoderm induction, stromal specification and maturation was performed according to an hPL-based protocol 37 (Fig. S6a, Fig.…”

“…The adult foreskin fibroblast-derived hiPSC line BIHi001-A was registered and described in detail at https://hpscreg.eu/cell-line/BIHi001-A. Adult bone marrow-and neonatal umbilical cord blood-derived hiPSCs are described elsewhere37 . All hiPSC strains used in this study were created by non-integrating Sendai virus-based reprogramming.…”

“…All cells were cultured in humidified nitrogencontrolled incubators at 37°C, 5% O2 and 5% CO2, and were large scale expanded in four-layered cell factories (CF-4, 140360, Nunc/Thermo). For selected animal experiments, fibroblasts were cultured for comparison in α-MEM/10% FBS, otherwise supplemented as described above, whenever specified in the results section.Human iPSCs generation and differentiationHuman iPSCs were reprogrammed from primary stromal cells derived from bone marrow and umbilical cord blood using a non-integrative Sendai viral vector kit (A1378001, Life Technologies) at the Harvard Stem Cell Institute (HSCI) iPSC Core Facility (Cambridge, MA, USA) adapted from an established protocol49 and characterized in a previous project37 . In addition, we used hiPSCs from dermal fibroblasts (BIHi001-A), generated and characterized by the Stem Cell Core Facility, Charité, Berlin Institute of Health.…”

Improving Human Induced Pluripotent Stem Cell-Derived Megakaryocyte Differentiation and Platelet Production

“…We focused on platelet production due to our interest in the regenerative potential of human platelets [ 26 ]. We observed recently that mesoderm induction in advance of subsequent lineage specification was beneficial for several middle germ layer-derived cell types including stromal cells [ 27 ] and vascular endothelial cells [ 28 ]. We included activin A [ 29 ], BMP-4, CHIR, and VEGF as an initial two-day mesoderm induction step in our protocol.…”