Development of Induced Pluripotent Stem Cell-based Therapies for Hematopoietic Stem Cell Disorders

Joseph A. Panos, Luigi J. Alvarado Ph.D., André Larochelle M.D. Ph.D. National Heart Lung and Blood Institute, Hematology Branch, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892

Mouse Transplantation. Immune-deficient (NSG) mice were injected i.v. with ~1x106 human CD34+ cells or iPSC-derived HSPCs immediately after a 4.5 hour-pre-culture at 39.5°C or 37°C. Twenty-four hours post-injection, mice were euthanized, their BM harvested from 6 bones, stained with anti-human CD45-FITC antibodies, and analyzed by flow cytometry. Confocal Microscopy. For visualization of membrane domains, cells were stained with AlexaFluor 555-conjugated Cholera toxin subunit B and visualized with a Zeiss LSM 780 confocal microscope.

Hematopoietic stem cells (HSCs) have the remarkable ability to reconstitute and maintain a functional hematopoietic system for the lifespan of an individual. In patients with inherited bone marrow (BM) failure disorders, HSCs are insufficient. Allogeneic HSC transplantation offers a potential cure, but there are well-described limitations. Also, sufficient autologous HSCs for gene therapy applications are unavailable in these patients, and methodologies for expansion of these rare HSCs are inexistent. Induced pluripotent stem cell (iPSC)-based therapies are a tractable alternative for these patients because of their potential to provide an unlimited source of autologous cells for gene therapy applications. Several protocols have been developed to generate hematopoietic stem and progenitor cells (HSPCs) from iPSCs but they are inefficient at producing the quantity and quality of HSCs required for clinical applications. Namely, phenotypically defined HSCs derived from iPSCs are incapable of reconstituting the hematopoietic system long-term in animal models; the cause of this functional defect remains unclear. We have recently shown that homing and engraftment of HSPCs to their BM microenvironment is mediated by polarized membrane domains enriched in adhesion molecules. The presence of polarized membrane domains on iPSC-derived HSCs has never been investigated as a possible cause for the homing/engraftment defect. A recent study has suggested that brief exposure of native HSPCs to hyperthermic conditions enhances domain polarization, homing, and engraftment.

There are no relevant conflicts of interest to disclose.

21 days post-transplant

Introduction

Objectives

Methods

Results

Conclusions

Hyperthermic treatment of human CD34+ cells before transplantation enhances their BM homing potential

Summary Impact of iPSC plating density on HSPC differentiation

1.  The protocol optimized in this study provides an efficient approach for the generation of human HSPCs with a primitive CD45lo/CD34hi phenotype from iPSCs generated from mobilized CD34+CD38- cells.

2.  Initial plating density of iPSCs was identified as a critical factor for efficient hematopoietic differentiation, with 70,000 iPSCs as the most efficient cell density for generation of CD34hi/CD45lo progenitors with the iPSC line utilized.

3.  Days 10-14 of the differentiation protocol were identified as optimal collection times for HSPCs.

Hyperthermic Treatment and HSPC Homing

Results

Human iPSCs were reprogrammed using Sendai viral vectors from normal G-CSF-mobilized CD34+/CD38- cells. iPSC colonies were cultured on Matrigel-coated plates in E8 medium. Cells were exposed to a cocktail of mesodermal differentiation factors for 3 days in APEL medium, followed by hematopoietic differentiation for 10 days. iPSC-differentiated cells were harvested from the culture supernatant starting at Day 10.

Methods

Results Hematopoietic Differentiation from Human iPSCs

Time course depiction of HSPC generation from iPSCs Exposure to mesodermal and hematopoietic differentiation factors promoted the formation of a hemogenic endothelial monolayer. As early as Day 7, spherical hematopoietic-like cells formed superficial clusters over the endothelial monolayer. These cells spontaneously sprouted in the culture supernatant between Days 10 and 14, allowing easy harvest.

Optimization of iPSC plating density The initial iPSC plating density was identified to be critical for optimal hematopoietic differentiation from human iPSCs.

Homing of human CD34+ cells was measured as the percentage of human CD45+ cells in the mouse BM 24 hours after transplantation. The proportion of CD45+ cells was significantly greater in the heat-treated group compared to the heat-untreated group.

Future Directions 1.  RNA-sequencing assays will be performed to assess the

genetic similarities between iPSC-derived HSCs and bona fide human HSCs.

2.  Functional homing experiments using iPSC-derived HSCs will be performed with adjuvants, such as a mild hyperthermic treatment, to assess the potential capacity of iPSCs to serve as a viable source of HSCs for bone marrow failure transplantation therapies.

References [1] Focosi, D., et al. Blood Cancer J., 2014. [2] Kaufman, D.S. Blood, 2009.

Generation of human HSPCs (CD45+CD34+) from iPSCs at various iPSC cell density Heat-treatment promotes the polarization of lipid rafts.

Density (cells/plate)

CD34+/CD45+ count

CD34lo/CD45hi count

CD34hi/CD45lo count

CD34hi /CD45lo -CD34lo /CD45hi

Ratio

Efficiency of HSPC

generation

70,000 151,438 15,003 30,439 2.03 6.49

90,000 17,523 819 5,291 6.56 1.71

100,000 50,607 1,701 15,485 9.10 1.51

Estimation of various HSPC counts shows that the density condition of 70,000 cells per plate most efficiently generated the desired CD34hi/CD45lo progenitor population for harvests conducted at Day 12.

[3] Capitano, M., et al. Stem Cells, 2015. [4] Larochelle, A. Blood, 2012.

1.  Develop a scalable protocol that supports hematopoietic differentiation of human iPSCs to meet the threshold cell dose of HSPCs for clinical transplantation.

2.  Investigate the presence of polarized membrane domains on native and iPSC-derived HSPCs, and the potential role of hyperthermia to enhance domain polarization, HSPC homing, and engraftment.

CD34hi/CD45lo (primitive HSPCs) and CD34lo/CD45hi (more mature HSPCs) populations, were observed in supernatant collected from all cultures with initial iPSC plating densities >70,000 cells per plate.

70,000 iPSCs 90,000 iPSCs 100,000 iPSCs

Hematopoietic Differentiation from Human iPSCs

Hyperthermic Treatment and HSPC Homing

Negative control 37oC treatment 39.5oC treatment

Hyperthermic Treatment and Domain Polarization

Lipid rafts on heat-treated and heat-untreated cells were detected directly using AlexaFluor 555-conjugated Cholera toxin subunit B, which binds specifically to ganglioside GM1. The polarization of lipid rafts, as a result of heat treatment, may explain enhanced CD34+ homing to the bone marrow niche.

Hyperthermic Treatment and HSPC Homing

Hematopoietic Differentiation from Human iPSCs

1.  Brief hyperthermic treatment of 39.5°C of native human CD34+ cells is effective in increasing their homing within the BM of NSG mice.

2.  Enhanced homing may be related to increased polarization of membrane-domain, enriched in adhesion molecules.

3.  These findings provide proof-of-principle that this approach may enhance homing and engraftment of iPSC-derived HSPCs.