Ex-vivo Stem Cell Expansion and Directed Differentiation

Founded in 2015 with licensed technology developed and studied for over two decades at Fred Hutchinson Cancer Research Center, Nohla’s proprietary platform technology is based on the density-dependent Notch ligand platform, a flexible platform that enables ex vivo expansion and directed differentiation of hematopoietic stem and progenitor cells. These cells are derived from umbilical cord blood and available for immediate use with no requirement for HLA matching.

Using purified CD34+ cord blood progenitor cells cultured in the presence of an engineered form of the Delta1ext -IgG ligand (DXI), the company is developing product candidates that can provide rapid, temporary myeloid engraftment for prevention of infection and facilitation of hematopoietic recovery following high dose chemotherapy.

Hematopoietic Stem/Progenitor Cell Expansion

The role of Notch in Hematopoiesis

In the early 1990’s Nohla Scientific Founder Dr. Irwin Bernstein at the Fred Hutchinson Cancer Research Center postulated that, as in other developing systems, hematopoietic stem cell-fate specification resulted from intercellular interactions with adjacent cells and was modulated by several families of molecules, including the Notch gene family. At that time, the Notch pathway was particularly well-studied in invertebrate systems with clear evidence that Notch played an important role in mediating intercellular interactions affecting cell-fate decisions within the central nervous system, eye, mesoderm, and ovaries.

A role for Notch in hematopoiesis was then further suggested by Dr. Bernstein’s detection of the human Notch1 gene in CD34+ or CD34+lin− human hematopoietic precursors. Subsequently, primary murine hematopoietic stem cells (HSC) were transduced with a retrovirus leading to constitutively active expression of the intracellular domain (ICD) of Notch1 and led to the emergence of an immortalized pluripotent cytokine-dependent cell line capable of both lymphoid and myeloid repopulation in vivo, thereby demonstrating a role for Notch in hematopoietic stem progenitor cell (HSPC) self-renewal. Although at the time a biological function for Notch in HSPC was not determined, this work suggested that manipulation of the Notch signaling pathway ex vivo in primary HSC could prove to be a novel approach for expanding HSPC.

Delta1 Ligand

To avoid the potential safety concerns of retroviral transduction, it was decided to activate endogenous Notch signaling in primary HSPC during culture. To that end, engineered Notch ligands were developed consisting of the extracellular domain (ECD) of the Notch ligands Jagged1 and Delta1. Notch ligands activate their receptors by physically pulling on the ECD in vivo; thus immobilization of the ligand proved necessary to activate endogenous Notch signaling in vitro. Using an immobilized form of the Notch ligand Delta1, Dr. Bernstein’s group was able to sufficiently activate endogenous receptors and induce expansion of murine stem progenitor cells capable of in vivo reconstitution similar to that seen in the retroviral-mediated Notch overexpression experiments. In fact, culture of murine hematopoietic precursors with the immobilized ligand Delta1 and cytokines resulted in a several-log increase in progenitors capable of short-term lymphoid and myeloid repopulation.

Cord Blood Expansion

Given the clinical need for large numbers of hematopoietic progenitor cells capable of providing rapid myeloid recovery in vivo, especially in patients undergoing cord blood transplant (CBT), Nohla Scientific Founder and Chief Medical Officer Dr. Colleen Delaney extended the ex vivo expansion platform from murine to human HSPC where she noted a unique response of cord blood (CB) HSPC to Notch ligands as compared to CD34+ bone marrow cells or mobilized peripheral blood stem cells. She found that culture of isolated CB CD34+ HSPC in the presence of an engineered form of Delta1 ligand in serum free media supplemented with cytokines led to a greater than 2-log increase in the number of CD34+ cells and nearly a 16-fold increase in NOD/SCID mouse repopulating cell frequency compared to control.

The ability of these human cells to rapidly reconstitute (as early as 10 days post infusion) the myeloid compartment in immunodeficient mice indicated their potential clinical utility. In contrast to the studies using primary murine HSPCs, in vivo persistence of transplanted cells at 9 weeks and secondary transplantation studies suggested the presence of both long-term and short-term repopulating cells following culture of human CB progenitor cells on Delta ligand.

A key aspect of these studies was determination of whether the magnitude of Notch signaling played a role in the optimal generation of repopulating cells. Murine studies showed that the relatively lower amount of Notch signaling induced in cells cultured with lower densities of Delta-1 led to self-renewal of progenitors with primarily B-lymphoid and myeloid potential, whereas higher amounts of Notch signaling inhibited B cell differentiation and promoted differentiation towards the T cell lineage.

Concurrent studies performed with human HSPC also revealed important ligand dose-dependent effects whereby relatively lower densities of immobilized ligand substantially enhanced generation of NOD/SCID repopulating cells, with higher ligand densities promoting differentiation towards the T-cell lineage at the expense of repopulating cells.

Scientific Publications

Delaney C, Milano F, Cicconi L, Othus M, Becker PS, Sandhu V, Nicoud I, Dahlberg A, Bernstein IDB, Appelbaum FR, Estey EH. Infusion of a non-HLA-matched ex-vivo expanded cord blood progenitor cell product after intensive acute myeloid leukaemia chemotherapy: a phase 1 trial. Lancet Haematol. 2016 Jul;3(7):e330-9. Epub 2016 Jun 7

Milano F, Gooley T, Wood B, Woolfrey A, Flowers ME, Doney K, Witherspoon R, Mielcarek M, Deeg JH, Sorror M, Dahlberg A, Sandmaier BM,Salit R, Petersdorf E, Appelbaum FR, Delaney C. Cord-Blood Transplantation in Patients with Minimal Residual Disease. N Engl J Med. 2016 Sep 8;375(10):944-53.

Li J, Blake J, Delaney C. Identificationof CD137+ IFN-g- CD4+ Alloreactive T Cells Post Myeloablative Double Cord BloodTransplant: A Synergistic Role in the Graft Versus Graft Interaction. ASBMT AnnualMeeting, San Diego, CA February 2015.

Dahlberg A,Milano F, Delaney C. Infusion of ex vivo expanded cord blood progenitor cells is associated with reduced hospital daysand utilization of opiate infusion and total parental nutrition in pediatricpatients receiving myeloablative cord blood transplantation. ASBMT AnnualMeeting, San Diego, CA February 2015.


Delaney C, Milano F, Cicconi L, Othus M, Becker PS, Sandhu V, Nicoud I, Dahlberg A, Bernstein IDB, Appelbaum FR, Estey EH. Infusion of a non-HLA-matched ex-vivo expanded cord blood progenitor cell product after intensive acute myeloid leukaemia chemotherapy: a phase 1 trial. Lancet Haematol. 2016 Jul;3(7):e330-9. Epub 2016 Jun 7

Milano F, Gooley T, Wood B, Woolfrey A, Flowers ME, Doney K, Witherspoon R, Mielcarek M, Deeg JH, Sorror M, Dahlberg A, Sandmaier BM,Salit R, Petersdorf E, Appelbaum FR, Delaney C. Cord-Blood Transplantation in Patients with Minimal Residual Disease. N Engl J Med. 2016 Sep 8;375(10):944-53.

Milano F, Heimfeld S, Gooley T, Jinneman J, Nicoud I, Delaney C. Correlation of Infused CD3(+)CD8(+) Cells with Single Donor Dominance after Double-Unit Cord Blood Transplantation. Biol Blood Marrow Transplant 2013 Jan: 19(1):156-60.

Newell LF, Milano F, Nicoud IB, Pereira A, Gooley TA, Heimfeld S, Delaney C. Early CD3 peripheral blood chimerism predicts the long-term engrafting unit following myeloablative double cord blood transplantation. BBMT. 2012 Aug;18(8):1243-9.

Gutman JA, Turtle CJ, Manley TJ, Heimfeld S, Bernstein ID, Riddell SR, Delaney C. Single-unit Dominance after Double-Unit Umbilical Cord Blood Transplantation Coincides with a Specific CD8+ T-Cell Response Against theNonengrafted Unit (Plenary Paper) Bood 2010 Jan 28;115(4):757-65.

Brustein C, Gutman J, Weisdorf C, Woolfrey A, DeFor T, Gooley T, Verneris M, Appelbaum F, Wagner J, Delaney C. Allogeneic Hematopoietic Cell Transplantation for Hematolotic Malignancy:Relative Risks and Benefirs of Double Umbilical CordBlood. Blood 2010 Nov 25:(116922):4693-9.

Delaney C, Heimfeld S,Brashem-Stein C, Voorhies H, Manger R and Bernstein ID. Notch-mediated expansion of human cord blood progenitor cells capableof rapid myeloid reconstitution. Nature Medicine, 2010 Feb;16(2):232-6.

Dahlberg A, Woo S, Delaney C, Boyle P, Gnirke A, Bock C, Bernstein BE, Meissner A, Gottardo R, Bernstein ID. Notch-mediated expansion of cord blood progenitors: maintenance of transcriptional and epigenetic fidelity. Leukemia. 2015 Sep; 29(9):1948-51

Hadland BK, Varnum-Finney B, Poulos MG, Moon RT, Butler JM, Rafii S, Bernstein ID. Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros-derivedhematopoietic stem cells. J Clin Invest. 2015 May; 125(5):2032-45.

Dahlberg A, Brasheim-Stein C, Delaney C, Bernstein ID. Enhanced Generation of Cord Blood Hematopoietic Stem and Progenitor Cells by Culture with Stem Regenin1 and Delta1(ext-IgG). Leukemia 2014 Oct 28 (10):2097-101.

Blake JM, Nicoud IB, Weber D,Voorhies H, Guthrie KA, Heimfeld S, Delaney C. Improved immunomagnetic enrichment of CD34 (+) cells from umbilicalcord blood using the CliniMACS cell separation system. Cytotherapy. 2012 Aug; 14(7):818-22.

Bernstein I, Delaney C. Engineering Stem Cell Expansion. Cell Stem Cell. 10, 113 (2012).

Dahlberg A, Delaney C, Bernstein ID. Ex Vivo Expansion of Human Hematopoietic Stem and Progenitor Cells. Blood, 2011 Jun 9; 117(23):6083-90.

Varnum-Finney B, Halasz LM, Sun M, Gridley T, Radtke F, Bernstein ID. Notch2 governs the rate of generation of mouse long- and short-term repopulating stem cells. J Clin Invest. 2011 Mar;121(3):1207-16.

Varnum-Finney B,Dallas MH, Kato K, Bernstein ID. Notchtarget Hes5 ensures appropriate Notch induced T- versus B-cell choices in thethymus. Blood. 2008 Mar 1;111(5):2615-20.

Dallas MH, Varnum-Finney B, Martin PJ, Bernstein ID. Enhanced T-cell reconstitution byhematopoietic progenitors expanded ex vivo using the Notch ligand Delta1 Blood. 2007 Apr 15;109(8):3579-87.

Aoyama K, Delaney C,Varnum-Finney B, Kohn AD, Moon RT, Bernstein ID. The interaction of the Wnt and Notch pathways modulates natural killerversus T cell differentiation. Stem Cells. 2007 Oct; 25(10):2488-97.

Delaney C, Varnum-Finney B,Aoyama K, Brashem-Stein C, Bernstein ID. Dose-dependent effects of the Notch ligand Delta1 on marrow repopulating ability of cord blood cells. Blood. 106(8):2693-9, Oct 2005.

Dallas MH, Varnum-Finney B, Delaney C, Kato K, Bernstein ID. Density of the Notchligand Delta1 determines generation of B and T cell precursors fromhematopoietic stem cells. J Exp Med. 201(9):1361-6, 2005

Varnum-Finney B,Brashem-Stein C, Bernstein ID. Combined effects of Notch signaling andcytokines induce a multiple log increase in precursors with lymphoid andmyeloid reconstituting ability. Blood, 101: 1784-1789, 2003.

Ohishi K1, Varnum-Finney B, Bernstein ID. Delta-1 enhances marrow and thymusrepopulating ability of human CD34(+) CD38(-) cordblood cells. J Clin Invest. 2002 Oct;110(8):1165-74.

Ohishi K1, Varnum-Finney B, Serda RE, Anasetti C, Bernstein ID. The Notch ligand, Delta-1, inhibits the differentiation of monocytesinto macrophages but permits their differentiation into dendritic cells. Blood. 2001 Sep 1;98(5):1402-7.

Ohishi K1, Varnum-Finney B, Flowers D, Anasetti C, Myerson D, Bernstein ID. Monocytes express high amounts of Notch and undergo cytokine specificapoptosis following interaction with the Notch ligand, Delta-1. Blood. 2000May 1;95(9):2847-54.

Varnum-Finney B, Wu L,Yu M, Brashem-Stein C, Staats S, Flowers D, Griffin JD, Bernstein ID. Immobilization of Notch ligand Delta-1 is required for induction of Notch signaling. J Cell Sci 2000 113:4313-4318.

Varnum-Finney B, Xu L, Brashem-Stein C, Nourigat C, Flowers D, Bakkour S,Pear WS, and Bernstein ID. Pluripotent, ctyokine dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nature Med6:1278-1281, 2000.

Varnum-Finney B, Purton LE, Yu M, Brashem-Stein C, Flowers D, Staats S, Moore KA, Le Roux I, Mann R, Gray G, Artavanis-Tsakonas S, Bernstein ID. The Notch ligand,Jagged-1, influences the development of primitive hematopoietic precursor cells. Blood. 1998 Jun 1;91(11):4084-91.

Milner LA, Kopan R, Martin DI, Bernstein ID. A human homologue of the Drosophila developmental gene, Notch, is expressed in CD34+ hematopoietic precursors. Blood. 1994 Apr 15;83(8):2057-62.