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Hematopoietic Stem Cells and Blood Cells

Large-scale production of megakaryocytes in microcarrier supported stirred suspension bioreactors

Eicke D., Baigger A., Schulze K., Latham S.L., Halloin C., Zweigerdt R., Guzman C.A., Blasczyk R., Figueiredo C.
Scientific reports, 2018

Although extensive research has been dedicated to developing processes for differentiating PLTs from MKs, no sustainable process for large-scale MK production is available. This study aimed at developing an effective, xeno-free and scalable system to produce high numbers of MKs. In particular, microcarrier beads-assisted stirred bioreactors were evaluated as a means of improving MK yields. This method resulted in the production of 18.7 Å~ 107 MKs per 50 ml medium. Biolaminin 521-coated microcarriers increased MK production per iPSC by up to 10-fold. MKs obtained in this system showed typical features of mature MKs and were able to produce PLTs in vitro and in vivo. To increase safety, MKs produced in the bioreactors were irradiated; a procedure that did not affect their capability to form proPLTs and PTLs after transfusion.


Superior Red Blood Cell Generation from Human Pluripotent Stem Cells Through a Novel Microcarrier-Based Embryoid Body Platform

Sivalingam J., Lam A.T., Chen H.Y., Yang B.X., Chen A.K., Reuveny S., Loh Y.H, Oh S.K.

Conventional methods for hematopoietic differentiation of human pluripotent stem cells (hPSC) rely on embryoid body (EB) formation and/or co-culture with xenogeneic cell lines. In this study, the authors describe the development of a scalable, serum-free, xeno-free, and chemically defined microcarrier-based platform using human recombinant laminin-521 as an extracellular matrix (ECM) for hPSC expansion, EB formation, and subsequently hematopoietic differentiation of hPSC to red blood cells (RBS). Improved survival and better quality EBs generated with the microcarrier-based method resulted in significantly improved mesoderm induction and, when combined with hematopoietic differentiation, resulted in at least a 6-fold improvement in hematopoietic precursor expansion, potentially culminating in an 80-fold improvement in the yield of RBS generation compared to a conventional EB-based differentiation method. In addition, they show efficient terminal maturation and generation of mature enucleated RBCs using a co-culture system that comprised primary human mesenchymal stromal cells.


Generation of HLA-Universal iPSC-Derived Megakaryocytes and Platelets for Survival Under Refractoriness Conditions

Börger A-K., Eicke D., Wolf C., Gras C., Aufderbeck S., Schulze K., Engels L., Eiz-Vesper B., Schambach A., Guzman C.A., Lachmann N., Moritz T., Martin U., Blasczyk R., Figueiredo C.
Molecular Medicine, 2016

Here, the authors have established a protocol for the generation of megakaryocytes (MK) and platelets (PLTs) from an HLA-universal and virtually unlimited cell source (iPSCs) under xeno-free and defined conditions to facilitate their future translation into clinical application. HLA-universal iPSC was generated and cultured on laminin-521. The expression of HLA class I was silenced (shRNA) by up to 82% and remained stable during iPSC cultivation. The generation of MK and PLTs from an HLA-universal iPSC were conducted on laminin-521. On d19, differentiation rates of MKs and PLTs with means of 58% and 76% were observed, respectively. HLA-universal iPSC-derived MKs showed polyploidy with DNA contents higher than 4n and formed proPLTs. Importantly, differentiated MKs remained silenced for HLA class I expression. HLA-universal MKs produced functional PLTs. Notably, iPSC-derived HLA-universal MKs were capable to escape antibody-mediated complement- and cellular-dependent cytotoxicity. Furthermore, HLA-universal MKs were able to produce PLTs after in vivo transfusion in a mouse model indicating that they might be used as an alternative to PLT transfusion. 


Integrin α6β1 Is the Main Receptor for Vascular Laminins and Plays a Role in Platelet Adhesion, Activation, and Arterial Thrombosis

Schaff et al.
Circulation. 2013

Here, the authors show that laminin-411, laminin-511, and laminin-521, but not laminin-211, allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin α6β1. Platelets from integrin α6 knockout mice failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. α6β1-Deficient mice presented a marked decrease in arterial thrombosis in 3 models of injury of the carotid, aorta, and mesenteric arterioles. The authors thus reveal an unsuspected important contribution of laminins to thrombus formation in vivo.


Characterization and functional analysis of laminin isoforms in human bone marrow

Siler U., Seiffert M., Puch S., Richards A., Torok-Storb B., Müller C.A, Sorokin L., Klein G.
Blood, 2000

Based on gene expression, laminin-411/421 and laminin-511/521 are the most abundant laminin isoforms synthesized by human bone marrow stromal cells. Laminin-511/521 preparations showed strong adhesive interactions with human CD34+ cell lines. Antibodies against the B1 integrin subunit inhibited these interactions. In addition to its adhesion-mediating properties, laminin-511/521 preparations also showed a mitogenic activity for human hematopoietic progenitor cells. Other laminin isoforms tested, especially laminin-111 and laminin-211 and 221, showed only weak or no adhesive interactions with the hematopoietic cell lines tested and are suggested to play a minor role in the hematopoietic microenvironment. In the bone marrow, LN‐511 and 521 are the major laminins and show strongly adhesive and mitogenic activities toward early developing HSCs.


Contribution of α6 integrins to hematopoietic stem and progenitor cell homing to bone marrow and collaboration with α4 integrins

Qian H., Tryggvason K., Jacobsen S.E., Ekblom M.
Blood, 2006

The integrin a6 chain is ubiquitously expressed in human and mouse hematopoietic stem and progenitor cells. The integrin a6 chain is ubiquitously expressed in human HPCs. Laminin-411 and -511, are present in subendothelial basement membranes of sinusoids in bone marrow, at sites of hematopoietic cell development and trafficking and might, therefore, regulate HSC functions. In this paper, they show that mouse HSC and progenitors express a6B1 integrin which mediates high cell adhesion to laminin-511 and 521 and to laminin-411 to a lower extent. Blocking of a6 significantly reduced progenitor cell homing to bone marrow in mice. Integrin a4 receptors are also important for homing of HSCs to bone marrow (but not to the spleen). The first data showing that a6 integrins (LN521/511 binding) function in vivo as hematopoietic stem and progenitor cell homing receptors. Also, show the role of integrin a4 receptor for homing of long-term multilineage reconstituting HSCs and collaboration of these 2 integrins in homing of short-term HSCs.


Laminin isoform-specific promotion of adhesion and migration of human bone marrow progenitor cells

Gu Y-C., Kortesmaa J., Tryggvason K., Persson J., Ekblom P., Jacobsen S-E., Ekblom M.

Here they studied human bone marrow cell adhesion to laminin-511/521, laminin-411, laminin-111, and fibronectin. About 35% to 40% of CD34+ and CD34+CD38- stem and progenitor cells adhered to laminin-511/521, and 45% to 50% adhered to fibronectin, whereas they adhered less to laminin-411 and laminin-111. Adhesion of CD34+CD38- cells to laminin-511/521 was maximal without integrin activation, whereas adhesion to other proteins was dependent on protein kinase C activation. Integrin a6 chain expressed on most CD34+ and CD34+CD38-cells. Laminin-511/521 was highly adhesive to lineage-committed myelomonocytic and erythroid progenitor cells and most lymphoid and myeloid cell lines studied, whereas fibronectin and laminin-411 were less adhesive. Laminin-511/521was a ubiquitous adhesive protein for differentiated precursors of both B-lymphocytic, erythroid, megakaryocytic, and myelomonocytic cell lineages, whereas adhesion to laminin-411 and laminin-111 was restricted to a few cell lines. CD34+ cell migration was greatly enhanced through membranes coated with Laminin-511/521 and laminin-411. Our findings raise the possibility that a4 and a5 laminins are involved in mobilization and homing of hematopoietic progenitor cells.


Laminin expression during bone marrow mononuclear cell transplantation in hepatectomized rats

Carvalho S., Cortez E., Stumbo A.C., Thole A., Caetano C., Marques R., Pelajo-Machado M., Cristóvao Porto L., Carvalho L.
Cell Biology International, 2008

The aim of this study was to investigate laminin expression during liver regeneration induced by partial hepatectomy followed by bone marrow mononuclear cell (BMMNC) transplantation (injected into recently hepatectomyzed rats via the portal vein). Results showed that 15 min after partial hepatectomy, a transplanted CD34+ HSC was found in contact with laminin, which was localized in the portal and centrilobular veins of rat livers. Furthermore, 1 and 3 days after hepatectomy, transplanted BMMNCs were found in the hepatic sinusoids (endothelia) expressing laminin. These results strongly suggest that laminin might be an important extracellular matrix component for bone marrow cell attachment and migration in the injured liver.


Release of Matrix Metalloproteinase-8 During Physiological Trafficking and Induced Mobilization of Human Hematopoietic Stem Cells

Steinl C., Essl M., Schreiber T.D., Geiger K., Prokop L., Stevanovic´ S., Pötz O., Abele H., Wessels J.T, Aicher W.K., Klein G.
Stem Cells Dev., 2013

In the present study, we provide evidence that the collagenase MMP-8 is involved in stem cell mobilization. Cell–matrix adhesion assays were performed with LM-511 where MACS-isolated CD34+ cells were allowed to attach to immobilized LM-511. A rapid release of MMP-8 from isolated neutrophil granulocytes can be observed during an in vitro culture. Activated MMP-8 degrades LM-511 but without influencing the HSC progenitor–matrix adhesion. This since MMP-8 strongly digests the LM-a5 chain but leaves the LG1–3 domains intact.


Characterization of bone marrow laminins and identification of alpha5-containing laminins as adhesive proteins for multipotent hematopoietic FDCP-Mix cells

Gu Y., Sorokin L., Durbeej M., Hjalt T., Jonsson J.I., Ekblom M.
Blood, 1999

Laminins interact in vitro with mature blood cells and malignant hematopoietic cells. Here they show that laminins are widely expressed in mouse bone marrow. Laminin a2, a4, and a5 polypeptides were found suggesting the presence of laminin-211, laminin-411, and laminin-511 in the bone marrow. Gene expression of laminin a1, a2, a4, and a5 chains in long-term bone marrow cultures, indicating up-regulation of laminin a1 expression in vitro. Laminins containing a5 chain, in contrast to laminin-1, were strongly adhesive for multipotent hematopoietic FDCPmix cells. Integrin a6 and b1 chains mediated this adhesion.